Elastomeric Proteins

ABSTRACT

This invention relates to elastomeric protein and elastomeric protein production. In particular, the invention is directed to elastomeric protein sequences, including methods and compositions for production of elastomeric protein sequences, such as expression constructs, and host cells, and including compositions generated from the elastomeric protein sequences.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/477,133, filed Jul. 10, 2019, which is a 371 national phaseapplication of International Application No. PCT/US18/13839, filed Jan.16, 2018, which claims the benefit of U.S. Provisional Application No.62/446,230, filed Jan. 13, 2017, the entire disclosure of which ishereby incorporated by reference, in its entirety, for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 31, 2021, isnamed BTT-007C1_CRF_sequencelisting.txt and is 167,578 bytes in size.

TECHNICAL FIELD

The present disclosure relates generally to elastomeric protein andelastomeric protein production. Specifically, the present disclosurerelates to elastomeric protein sequences, expression constructs, hostcells, and solids.

BACKGROUND

Elastomeric proteins are polypeptides that exhibit viscoelasticmechanical properties, and include elastin, resilin, abductin, andoctopus arterial elastomers. Resilin is a particularly interestingelastomeric protein because it dissipates very little energy duringloading and unloading. Resilin is found in many insects, and the lowenergy dissipation enables the extraordinary ability of many insectspecies to jump or pivot their wings very efficiently. The uniqueproperties of resilin make it an interesting elastomeric material thatcould have many industrial applications. However, resilin exists in onlyvery small quantities in nature, and therefore cannot becost-effectively farmed by raising insects.

Variations of natural resilins and resilin-like proteins (based onresilin sequences) have been recombinantly produced by a number ofgroups in E. coli cultures, and have been isolated by lysing the cellsto extract recombinantly expressed proteins, and using affinitychromatography techniques to purify (Elvin et al., 2005; Charati et al.;2009, McGann et al., 2013). The recombinantly produced resilin andresilin-like proteins have been cross-linked targeting the tyrosineresidues that also form the cross-links in natural resilin (see, forexample, Elvin et al., 2005; Qin et al., 2011). Recombinantly producedresilin has also been cross-linked targeting lysine residues (Li et al.,2011) or cysteine residues (McGann et al., 2013). Cross-linkedrecombinantly produced resilin and resilin-like proteins have shownmechanical properties similar to those of natural resilin, withresilience values in excess of 90% (Elvin et al., 2005, Qin et al.,2011, Li et al., 2011).

In one study, 70-80 mg of recombinant resilin-like protein were producedper liter of E. coli culture, and the resilin-like protein was purifiedby Ni-NTA affinity chromatography (Charati et al., 2009). More efficientexpression systems have been developed, which have produced 300-450 mg/Lof recombinant resilin-like proteins from E. coli host cells (Lyons, etal., 2009). More efficient methods have also been developed forpurifying the resilin-like proteins from lysed E. coli host cells, basedon salt precipitation followed by heating (Qin et al., 2011; Lyons etal., 2009). However, improved systems for expressing and purifyingelastomeric proteins (e.g., resilin and resilin-like proteins) withgreater productivity are desired to provide more efficient proteinproduction at larger scales.

At least one drawback of recovering expressed proteins by lysing cellsfollowed by simple precipitation-based purification techniques is thatthe resulting proteins tend to have low purity due to cellular proteinsfrom the lysed cells contaminating the target protein. Low purity canresult in a variety of product defects, including low resilience.Furthermore, intracellular accumulation of protein can lead to toxicityand therefore decreased efficiency of production of recombinantelastomeric proteins. What is needed, therefore, are improved methodsfor expression and purification of recombinant elastomeric proteins thatinclude methods to recover elastomeric proteins from extracellularportions. What is also needed are improved methods for expression andpurification of recombinant elastomeric proteins (e.g., resilin andresilin-like proteins) that have a greater production efficiency.

SUMMARY OF THE INVENTION

According to some embodiments, provided herein is a method for producinga composition comprising a recombinant resilin protein, the methodcomprising: culturing a population of recombinant host cells in afermentation, wherein said recombinant host cells comprise a vectorcomprising a secreted resilin coding sequence, and wherein saidrecombinant host cells secrete a recombinant resilin protein encoded bysaid secreted resilin coding sequence; and purifying said recombinantresilin protein from said fermentation.

In some embodiments, the recombinant resilin protein is a full-length ortruncated native resilin. In some embodiments, the native resilin isfrom an organism selected from the group consisting of: Drosophilasechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans,Ctenocephalides felis, Bombus terrestris, Tribolium castaneum, Apismellifera, Nasonia vitripennis, Pediculus humanus corporis, Anophelesgambiae, Glossina morsitans, Atta cephalotes, Anopheles darlingi,Acyrthosiphon pisum, Drosophila virilis, Drosophila erecta, Lutzomyialongipalpis, Rhodnius prolixus, Solenopsis invicta, Culexquinquefasciatus, Bactrocera cucurbitae, and Trichogramma pretiosum. Insome embodiments, the recombinant resilin protein comprises SEQ IDNO: 1. In some embodiments, the recombinant resilin protein comprisesSEQ ID NO: 4.

In some embodiments, the recombinant resilin protein comprises an alphamating factor secretion signal. In some embodiments, the recombinantresilin protein comprises a FLAG-tag. In some embodiments, the vectorcomprises more than one secreted resilin coding sequence.

In some embodiments, the recombinant host cells are yeast cells. In someembodiments, the yeast cells are methylotrophic yeast cells. In someembodiments, the recombinant host cells are a species selected from thegroup consisting of: Pichia (Komagataella) pastoris, Hansenulapolymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia(Scheffersomyces) stipitis, Pichia methanolica, Saccharomycescerevisiae, and Kluyveromyces lactis.

In some embodiments, the recombinant host cells produce the recombinantresilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.In some embodiments, the recombinant host cells produce a secretedfraction of the recombinant resilin that is greater than 50% as comparedto the total recombinant resilin protein expressed by the recombinanthost cells. In some embodiments, the recombinant host cells secrete therecombinant resilin at a rate of greater than 2 mg resilin/g dry cellweight/hour. In some embodiments, greater than 80% of the recombinantresilin is outside of the recombinant host cells in said fermentation.In some embodiments, the fermentation comprises at least 2 g recombinantresilin/L.

In some embodiments, purifying said recombinant resilin proteincomprises: generating a first pellet fraction and a first supernatantfraction by centrifuging the fermentation; and isolating the recombinantresilin protein from the first pellet fraction. In some embodiments,purifying said recombinant resilin protein further comprises: adding achaotrope to the first pellet fraction to generate a solution in whichthe recombinant resilin protein is soluble; generating a secondsupernatant fraction and a second pellet fraction by centrifuging thefirst pellet fraction comprising said chaotrope; and isolating thesoluble full-length resilin from the second supernatant fraction.

In some embodiments, provided herein is a vector comprising a secretedresilin coding sequence. In some embodiments, the secreted resilincoding sequence encodes a full-length or truncated native resilin. Insome embodiments, the secreted resilin coding sequence encodes amodified full-length or truncated native resilin. In some embodiments,the modified resilin comprises an addition, subtraction, replacement, orchange in position of an amino acid residue capable of cross-linking toanother resilin.

In some embodiments, the full-length or truncated native resilin is froman organism selected from the group consisting of: Drosophila sechellia,Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalidesfelis, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasoniavitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossinamorsitans, Atta cephalotes, Anopheles darlingi, Acyrthosiphon pisum,Drosophila virilis, Drosophila erecta, Lutzomyia longipalpis, Rhodniusprolixus, Solenopsis invicta, Culex quinquefasciatus, Bactroceracucurbitae, and Trichogramma pretiosum.

In some embodiments, the secreted resilin coding sequences encodes apolypeptide comprising SEQ ID NO: 1. In some embodiments, the secretedresilin coding sequence encodes a polypeptide comprising SEQ ID NO: 4.In some embodiments, the secreted resilin coding sequence encodes arecombinant resilin comprising one or more A-repeats or quasi-A-repeats.In some embodiments, the secreted resilin coding sequence encodes arecombinant resilin comprising one or more B-repeats or quasi-B-repeats.In some embodiments, the secreted resilin coding sequence encodes arecombinant resilin comprising either one or more A-repeats orquasi-A-repeats or one or more B-repeats or quasi-B repeats but notboth. In some embodiments, the secreted resilin coding sequence encodesa recombinant resilin comprising one or more A-repeats orquasi-A-repeats and one or more B-repeats or quasi-B-repeats.

In some embodiments, the recombinant resilin further comprises a chitinbinding domain. In some embodiments, the secreted resilin codingsequence encodes a polypeptide comprising an alpha mating factorsecretion signal. In some embodiments, the secreted resilin codingsequence comprises a FLAG-tag.

In some embodiments, the vector comprises more than one secreted resilincoding sequence. In some embodiments, the vector comprises 3 secretedresilin coding sequences. In some embodiments, the secreted resilincoding sequence is operatively linked to a constitutive or induciblepromoter.

Also provided herein, according to some embodiments, is a recombinanthost cell comprising one or more vectors comprising a secreted resilincoding sequence. In some embodiments, the recombinant host cell is ayeast cell. In some embodiments, the yeast cell is a methylotrophicyeast cell. In some embodiments, the recombinant host cell is a speciesselected from the group consisting of: Pichia (Komagataella) pastoris,Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia(Scheffersomyces) stipitis, Pichia methanolica, Saccharomycescerevisiae, and Kluyveromyces lactis.

In some embodiments, the recombinant host cell comprises 3 vectorscomprising a secreted resilin coding sequence.

In some embodiments, the recombinant host cell produces recombinantresilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.In some embodiments, the recombinant host cell has a secreted fractionof recombinant resilin that is greater than 50%. In some embodiments,the recombinant host cell secretes resilin at a rate of greater than 2mg resilin/g dry cell weight/hour.

Also provided herein, according to some embodiments, is a fermentationcomprising a recombinant host cell comprising one or more vectorscomprising a secreted resilin coding sequence and a culture mediumsuitable for growing the recombinant host cell.

In some embodiments, the fermentation comprises at least 2 g recombinantresilin/L.

In some embodiments of the fermentation, greater than 80% of recombinantresilin is outside of the recombinant host cells.

In some embodiments of the fermentation, the recombinant resilin isfull-length recombinant resilin.

Also provided herein, according to some embodiments, is a compositioncomprising recombinant resilin derived from a fermentation comprising arecombinant host cell comprising one or more vectors comprising asecreted resilin coding sequence and a culture medium suitable forgrowing the recombinant host cell. In some embodiments, the compositioncomprises at least 60% by weight of recombinant resilin.

In some embodiments, the composition has similar properties compared tocompositions comprising similar amounts of native resilins. In someembodiments, the composition has different properties compared tocompositions comprising similar amounts of native resilins.

In some embodiments, the composition comprises a resilience of greaterthan 50%. In some embodiments, the composition comprises has acompressive elastic modulus of less than 10 MPa. In some embodiments,the composition has a tensile elastic modulus of less than 10 MPa. Insome embodiments, the composition has a shear modulus of less than 1MPa. In some embodiments, the composition has an extension to break ofgreater than 1%. In some embodiments, the composition has a maximumtensile strength of greater than 0.1 kPa. In some embodiments, thecomposition has a Shore 00 Hardness of less than 90. In someembodiments, the composition comprises full-length resilin.

Also provided herein, according to some embodiments, is a method forproducing a composition comprising recombinant resilin, the methodcomprising the step of culturing a recombinant host cell comprising oneor more vectors comprising a secreted resilin coding sequence to producea fermentation under conditions that promote secretion of recombinantresilin from the recombinant host cell.

In some embodiments, the method for producing a composition comprisingrecombinant resilin further comprises the step of purifying saidrecombinant resilin to produce full-length native resilin. In someembodiments, purifying said recombinant resilin to produce full-lengthnative resilin comprises: generating a first pellet fraction and a firstsupernatant fraction by centrifuging the fermentation; and isolating therecombinant resilin protein from the first pellet fraction. In someembodiments, isolating the recombinant resilin protein from the firstpellet fraction comprises: adding a chaotrope to the first pelletfraction to generate a solution in which the recombinant resilin proteinis soluble; generating a second supernatant fraction and a second pelletfraction by centrifuging the first pellet fraction comprising saidchaotrope; and isolating the recombinant resilin protein from the secondsupernatant fraction.

In some embodiments, the method for producing a composition comprisingrecombinant resilin further comprises the step of cross-linking aplurality of said recombinant resilins. In some embodiments, saidcross-linking is enzymatic cross-linking. In some embodiments, saidcross-linking is photochemical cross-linking. In some embodiments, therecombinant resilin protein comprises a full-length resilin protein.

Also provided herein, according to some embodiments, is a fermentationcomprising a culture medium and a recombinant host cell, wherein therecombinant host cell comprises a vector, wherein the vector comprises asecreted resilin coding sequence, and wherein the recombinant host cellsecretes recombinant resilin at a rate of at least 2 mg/g dry cellweight/hour.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the structure of an exemplary resilin.

FIG. 2 is a flow diagram of methods for the production of compositionscomprising recombinant resilins.

FIG. 3 is an illustrative map of a vector that comprises 3 secretedresilin coding sequences.

FIG. 4A shows expression and secretion of 3× FLAG-tagged recombinantresilins in Pichia pastoris (Komagataella phaffii) recombinant hostcells as assayed by ELISA. FIG. 4B shows expression of recombinantresilins in Pichia pastoris (Komagataella phaffii) recombinant hostcells as assayed by Western blot (top; 3×FLAG-tagged recombinantresilins), and Coomassie (bottom; untagged recombinant resilins).

FIG. 5A shows productivities of recombinant host cells producingrecombinant resilins in rich media. FIG. 5B shows productivities ofrecombinant host cells producing recombinant resilins in minimal media.

FIG. 6 shows purification of 2 secreted recombinant resilins from 500 mLBMGY flask growth. For each sample, Lane 1 is the original supernatant;Lane 2 is the supernatant after precipitation; Lane 3 is the dialyzedprecipitate; Lane 4 is the heat-denatured proteins; and Lane 5 is thefinal purified recombinant resilin.

FIG. 7 shows photographs of proteinaceous block co-polymers comprisingcross-linked purified recombinant resilins in various shapes and forms.

FIG. 8 shows photographs of the compression of a proteinaceous blockco-polymer comprising cross-linked recombinant resilin.

FIG. 9 is an image of a gel showing the resulting bands from recombinantresilin compositions purified by selected methods described herein.

FIG. 10 is the full-length Drosophila sechellia resilin sequence(Ds_ACB) (SEQ ID NO: 10) that is expressed along with signal sequencesthat are later cleaved, according to some embodiments of the invention.

The figures depict various embodiments of the present disclosure forpurposes of illustration only. One skilled in the art will readilyrecognize from the following discussion that alternative embodiments ofthe structures and methods illustrated herein may be employed withoutdeparting from the principles described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure pertains.

The terms “a” and “an” and “the” and similar referents as used hereinrefer to both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context.

The term “about,” “approximately,” or “similar to” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which can depend in part on how the value ismeasured or determined, or on the limitations of the measurement system.It should be understood that all ranges and quantities described beloware approximations and are not intended to limit the invention. Whereranges and numbers are used these can be approximate to includestatistical ranges or measurement errors or variation. In someembodiments, for instance, measurements could be plus or minus 10%.

Amino acids can be referred to by their single-letter codes or by theirthree-letter codes. The single-letter codes, amino acid names, andthree-letter codes are as follows: G—Glycine (Gly), P—Proline (Pro),A—Alanine (Ala), V—Valine (Val), L—Leucine (Leu), I—Isoleucine (Ile),M—Methionine (Met), C—Cysteine (Cys), F—Phenylalanine (Phe), Y—Tyrosine(Tyr), W—Tryptophan (Trp), H—Histidine (His), K—Lysine (Lys), R—Arginine(Arg), Q—Glutamine (Gln), N—Asparagine (Asn), E—Glutamic Acid (Glu),D—Aspartic Acid (Asp), S—Serine (Ser), T—Threonine (Thr).

The terms “including,” “includes,” “having,” “has,” “with,” or variantsthereof are intended to be inclusive in a manner similar to the term“comprising”.

The term “microbe” as used herein refers to a microorganism, and refersto a unicellular organism. As used herein, the term includes allbacteria, all archaea, unicellular protista, unicellular animals,unicellular plants, unicellular fungi, unicellular algae, all protozoa,and all chromista.

The term “native” as used herein refers to compositions found in naturein their natural, unmodified state.

The terms “optional” or “optionally” mean that the feature or structuremay or may not be present, or that an event or circumstance may or maynot occur, and that the description includes instances where aparticular feature or structure is present and instances where thefeature or structure is absent, or instances where the event orcircumstance occurs and instances where the event or circumstance doesnot occur.

The term “secreted fraction” as used herein refers to the fraction ofrecombinant resilins that are secreted from cells compared to the totalresilins produced by the cells.

The term “secretion signal” as used herein refers to a short peptidethat when fused to a polypeptide mediates the secretion of thatpolypeptide from a cell.

The term “secreted resilin coding sequence” as used herein refers to anucleotide sequence that encodes a resilin as provided herein fused atits N-terminus to a secretion signal and optionally at its C-terminus toa tag peptide or polypeptide.

The term “recombinant” as used herein in reference to a polypeptide(e.g., resilin) refers to a polypeptide that is produced in arecombinant host cell, or to a polypeptide that is synthesized from arecombinant nucleic acid.

The term “recombinant host cell” as used herein refers to a host cellthat comprises a recombinant nucleic acid.

The term “recombinant nucleic acid” as used herein refers to a nucleicacid that is removed from its naturally occurring environment, or anucleic acid that is not associated with all or a portion of a nucleicacid abutting or proximal to the nucleic acid when it is found innature, or a nucleic acid that is operatively linked to a nucleic acidthat it is not linked to in nature, or a nucleic acid that does notoccur in nature, or a nucleic acid that contains a modification that isnot found in that nucleic acid in nature (e.g., insertion, deletion, orpoint mutation introduced artificially, e.g., by human intervention), ora nucleic acid that is integrated into a chromosome at a heterologoussite. The term includes cloned DNA isolates and nucleic acids thatcomprise chemically-synthesized nucleotide analog.

The term “vector” as used herein refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which generally refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated, but also includes linear double-stranded molecules such asthose resulting from amplification by the polymerase chain reaction(PCR) or from treatment of a circular plasmid with a restriction enzyme.Other vectors include bacteriophages, cosmids, bacterial artificialchromosomes (BAC), and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments can beligated into the viral genome. Certain vectors are capable of autonomousreplication in a cell into which they are introduced (e.g., vectorshaving an origin of replication that functions in the cell). Othervectors can be integrated into the genome of a cell upon introductioninto the cell, and are thereby replicated along with the cell genome.

The term “repeat” as used herein, in reference to an amino acid ornucleic acid sequence, refers to a sub-sequence that is present morethan once in a polynucleotide or polypeptide (e.g., a concatenatedsequence). A polynucleotide or polypeptide can have a direct repetitionof the repeat sequence without any intervening sequence, or can havenon-consecutive repetition of the repeat sequence with interveningsequences. The term “quasi-repeat” as used herein, in reference to aminoacid or nucleic acid sequences, is a sub-sequence that is inexactlyrepeated (i.e., wherein some portion of the quasi-repeat subsequence isvariable between quasi-repeats) across a polynucleotide or polypeptide.Repeating polypeptides and DNA molecules (or portions of polypeptides orDNA molecules) can be made up of either repeat sub-sequences (i.e.,exact repeats) or quasi-repeat sub-sequences (i.e., inexact repeats).

The term “native resilin” as used herein refers to an elastomericpolypeptide or protein produced by insects. GenBank Accession Nos. ofnon-limiting examples of native resilin includes the following NCBIsequence numbers: NP 995860 (Drosophila melanogaster), NP 611157(Drosophila melanogaster), Q9V7U0 (Drosophila melanogaster), AAS64829,AAF57953 (Drosophila melanogaster), XP 001817028 (Tribolium castaneum)and XP001947408 (Acyrthosiphon pisum).

The term “modified” as used herein refers to a protein or polypeptidesequence that differs in composition from a native protein orpolypeptide sequence, where the functional properties are preserved towithin 10% of the native protein or polypeptide properties. In someembodiments, the difference between the modified protein or polypeptideand the native protein or polypeptide can be in primary sequence (e.g.,one or more amino acids are removed, inserted or substituted) orpost-translation modifications (e.g., glycosylation, phosphorylation).Amino acid deletion refers to removal of one or more amino acids from aprotein. Amino acid insertion refers to one or more amino acid residuesbeing introduced in a protein or polypeptide. Amino acid insertions maycomprise N-terminal and/or C-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Amino acid substitutionincludes non-conservative or conservative substitution, whereconservative amino acid substitution tables are well known in the art(see for example Creighton (1984) Proteins. W. H. Freeman and Company(Eds)). In some embodiments, the modified protein or polypeptide and thenative protein or polypeptide amino acid or nucleotide sequence identityis at least 60%, at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, or at least 98% of the aminoacids or nucleotide bases.

The term “truncated” as used herein refers to a protein or polypeptidesequence that is shorter in length than a native protein or polypeptide.In some embodiments, the truncated protein or polypeptide can be greaterthan 10%, or greater than 20%, or greater than 30%, or greater than 40%,or greater than 50%, or greater than 60%, or greater than 70%, orgreater than 80%, or greater than 90% of the length of the nativeprotein or polypeptide.

The term “homolog” or “substantial similarity,” as used herein, whenreferring to a polypeptide, nucleic acid or fragment thereof, indicatesthat, when optimally aligned with appropriate amino acid or nucleotideinsertions or deletions with another amino acid or nucleic acid (or itscomplementary strand), there is amino acid or nucleotide sequenceidentity in at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98% ofthe amino acids or nucleotide bases, as measured by any well-knownalgorithm of sequence identity, such as FASTA, BLAST or Gap, asdiscussed above.

The term “resilin” as used herein refers to a protein or a polypeptide,capable of cross-linking to form an elastomer, where the protein orpolypeptide is a native resilin, or a native resilin that is modified,or a native resilin that is truncated. Resilins of the present inventionare preferably recombinant resilins. In some embodiments, recombinantresilins comprise a natural or modified (e.g., truncated orconcatenated) nucleotide sequence coding for resilin or resilinfragments (e.g., isolated from insects), heterologously expressed andsecreted from a host cell. In preferred embodiments, the secretedrecombinant resilin protein is collected from a solution extracellularto the host cell.

As used herein, the term “elastomer” refers to a polymer withviscoelasticity and typically weak inter-molecular forces (except forcovalent cross-links between molecules, if they are present).Viscoelasticity is a property of materials that exhibit both viscous andelastic characteristics when undergoing deformation, and thereforeexhibit time-dependent strain. Elasticity is associated with bondstretching along crystallographic planes in an ordered solid, andviscosity is the result of the diffusion of atoms or molecules inside anamorphous material. Elastomers that are viscoelastic, therefore,generally have low Young's modulus and high failure strain compared withother materials. Due to the viscous component of the material,viscoelastic materials dissipate energy when a load is applied and thenremoved. This phenomenon is observed as hysteresis in the stress-straincurve of viscoelastic materials. As a load is applied there is aparticular stress-strain curve, and as the load is removed thestress-strain curve upon unloading is different than that of the curveduring loading. The energy dissipated is the area between the loadingand unloading curves.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valueinclusively falling within the range, unless otherwise indicated herein,and each separate value is incorporated into the specification as if itwere individually recited herein.

DETAILED DESCRIPTION

Provided herein are compositions comprising recombinant resilins, andmethods for their production.

Resilins have many unique properties compared to petroleum-basedelastomers. Most notably, resilin has an extreme elastic efficiency,where very little of the energy input into deformation is lost as heat.Other desirable properties of resilin include, for example, desirableresilience, compressive elastic modulus, tensile elastic modulus, shearmodulus, extension to break, maximum tensile strength, hardness,rebound, and compression set. Moreover, resilin is a protein, andtherefore can be biodegraded, which makes it more environmentallyfriendly than petroleum-based polymers. Also, resilin is biocompatibleand can therefore be used in applications that involve contact withhumans or animals. Lastly, the mechanical properties of recombinantresilins can be tuned through varying protein sequence, proteinstructure, amount of intermolecular cross-linking and processingvariables to produce elastomers designed for a universe of specificapplications.

In some embodiments, the methods and compositions provided hereinprovide efficient means for producing large quantities of recombinantresilins. In some embodiments, large quantities of resilin andresilin-like polypeptides are obtained using recombinant host cells thatsecrete recombinant resilins via their secretory pathways. Suchsecretion of recombinant resilins a) avoids toxicity from intracellularaccumulation of recombinant resilins, b) simplifies purification byeliminating cell disruption or protein refolding processes, and c)provides opportunities for post-translational events (e.g., proteolyticmaturation, glycosylation, disulfide bond formation) that can modulatethe properties of the recombinant resilins.

Compositions Comprising Recombinant Resilins

In some embodiments, the compositions provided herein compriserecombinant resilins.

FIG. 1 illustrates an example of a native resilin, which contains anN-terminal A-domain comprising a plurality of repeat units comprisingthe consensus amino acid sequence YGXP (“A-repeat”), where X is anyamino acid; a chitin-binding type RR-2 (C) domain (Pfam referencePF00379; Rebers J E & Willis, J H. A conserved domain in anthropodcuticular proteins binds chitin. Insect Biochem Mol Biol 31:1083-1093);and a C-terminal B-domain comprising a plurality of repeat unitscomprising the consensus amino acid sequence UYZXZ (“B-repeat”), where Uis glycine or serine; Z is serine, glycine, arginine, or proline; and Xis any amino acid. Not all naturally occurring resilins have A-, C-, andB-domains. Native resilins produced by various insects typically haveinexact repeats (i.e., quasi-repeats) within the A- and/or B-domainswith some amino acid variation between the quasi-repeats.

In some embodiments, the recombinant resilins provided herein compriseone or more A-repeats. In some embodiments, the recombinant resilinscomprise N-terminal A-domains comprising a plurality of blocks ofA-repeat and/or quasi-A-repeat amino acid sub-sequences each with theconsensus sequence SXXYGXP, where S is serine, X is an amino acid, Y istyrosine, G is glycine, and P is proline.

In some embodiments, the recombinant resilins provided herein compriseone or more B-repeats. In some embodiments, the recombinant resilinscomprise a C-terminal B-domain comprising a plurality of blocks ofB-repeat and/or quasi-B-repeat amino acid sub-sequences each with theconsensus sequence GYZXZZX and/or SYZXZZX, where G is glycine; Y istyrosine; Z is serine, glycine, proline, or arginine; S is serine; and Xis any amino acid.

In some embodiments, the recombinant resilins provided herein compriseone or more A-repeats. In some such embodiments, the recombinantresilins comprise between 1 and 100 A-repeats, or from 2 to 50A-repeats, or from 5 to 50 A-repeats, or from 5 to 20 A-repeats.

In some embodiments, the recombinant resilins comprise one or moreconsensus sequences described by the formula,

(X₁-X₂-X₃-X₄)_(n)  (1)

wherein the brackets delineate a repeat or quasi-repeat of the consensussequence;wherein n describes the number of A-repeats or quasi-A-repeats, and isfrom 1 to 100, or from 2 to 50, or from 5 to 50, or from 5 to 20;wherein X₁ is a motif that is 4 amino acids in length, wherein the firstamino acid of X₁ is Y, and wherein the remaining amino acids of X₁ areGAP, GLP, GPP, GTP, or GVP;wherein X₂ is a motif that is from 3 to 20 amino acids in length;wherein X₂ comprises GGG, GGGG, N, NG, NN, NGN, NGNG, GQGG, GQGN, GQGQ,GQGQG, or 3 or more glycine residues, or wherein 50% or more of theresidues of X₂ are either glycine or asparagine, or wherein 60% or moreof the residues of X₂ are either glycine or asparagine, or wherein 70%or more of the residues of X₂ are either glycine or asparagine, orwherein 80% or more of the residues of X₂ are either glycine orasparagine;wherein X₃ is a motif that is from 2 to 6 amino acids in length, whereinX₃ is GG, LS, APS, GAG, GGG, KPS, RPS, or GGGG; and wherein X₄ is amotif that is from 1 to 2 amino acids in length, wherein X₄ is S, D, T,N, L, DS, DT, LS, SS, ST, TN, or TS.

In some such embodiments, the recombinant resilins comprise motifs X₁,X₂, X₃, and X₄ whereas in other embodiments, the recombinant resilinscomprise motifs X₁, X₂, X₃, or X₄, or combinations thereof.

In some embodiments, the recombinant resilins provided herein compriseone or more B-repeats. In some such embodiments, the recombinantresilins comprise between 1 and 100 B-repeats, or from 2 to 50A-repeats, or from 5 to 50 A-repeats, or from 5 to 20 A-repeats.

In some embodiments, the recombinant resilins comprise one or moreconsensus sequences described by the formula,

(X₁₁-X₁₂-X₁₃)_(m)  (2)

wherein the brackets delineate a repeat or quasi-repeat of the consensussequence;wherein m describes the number of B-repeats or quasi-B-repeats, and isfrom 1 to 100;wherein X₁₁ is a motif that is from 1 to 5 amino acids in length, thefirst amino acid is Y, and where the remaining amino acids can compriseGAP, GPP, SSG, or SGG;wherein X₁₂ is a motif that is from 2 to 5 amino acids in length andcomprises GQ, GN, RPG, RPGGQ, RPGGN, SSS, SKG, or SN; andwherein X₁₃ is a motif that is from 4 to 30 amino acids in length andcomprises GG, DLG, GFG, GGG, RDG, SGG, SSS, GGSF, GNGG, GGAGG, or 3 ormore glycine residues, or 30% or more of the residues are glycine, or40% or more of the residues are glycine, or 50% or more of the residuesare glycine, or 60% or more of the residues are glycine.

In some such embodiments, the recombinant resilins comprise motifs X₁₁,X₁₂, and X₁₃ whereas in other such embodiments, the recombinant resilinscomprise motifs X₁₁, X₁₂, or X₁₃, or combinations thereof.

In some embodiments, the recombinant resilins provided herein compriseone or more A-repeats, one or more B-repeats, and/or one or moreC-domain. In some embodiments, the recombinant resilins comprise one ormore A-repeats or one or more B-repeats but not both. In someembodiments, the recombinant resilins comprise one or more A-repeats butnot B-repeats or C-domains. In some embodiments, the recombinantresilins comprise one or more B-repeats but not A-repeats or C-domains.In embodiments in which the recombinant resilins comprise a C-domain,the C-domain can be situated either on the N-terminal or the C-terminalsides of the A-repeats or B-repeats, or between the A-repeats and theB-repeats.

In some embodiments, the recombinant resilins further comprise thesequence XXEPPVSYLPPS, where X is any amino acid. In some suchembodiments, the sequence is located on the N-terminal side of anA-repeat or B-repeat.

In some embodiments, the recombinant resilins are full-length nativeresilins expressed in a non-native environment. In some embodiments, therecombinant resilins comprise a truncated version of native resilins. Insome embodiments, the truncated native resilins comprise at least oneA-repeat. In some embodiments, the truncated native resilins comprise atleast one B-repeat. Non-limiting examples of full-length and truncatednative resilins are provided as SEQ ID NOs: 1 through 44. In someembodiments, the recombinant resilins are full-length Drosophilasechellia resilin (SEQ ID NO: 1). In some embodiments, the recombinantresilins are truncated Acromyrmex echinatior resilin (SEQ ID NO: 4). Insome embodiments, the recombinant resilins are full-length or truncatednative resilins that are cross-linked in a non-native manner (e.g., lessor more cross-linking, cross-linking via different amino acid residues).

In some embodiments, the recombinant resilins are modified full-lengthor truncated native resilins. In some embodiments, the recombinantresilins are at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%identical to a full-length or truncated native resilin. In someembodiments, the recombinant resilins are at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% identical to full-length Drosophila sechelliaresilin (SEQ ID NO: 1). In some embodiments, the recombinant resilinsare at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% identicalto truncated Acromyrmex echinatior resilin (SEQ ID NO: 4).

There are a number of different algorithms known in the art which can beused to measure nucleotide sequence or protein sequence identity. Forinstance, polynucleotide sequences can be compared using FASTA, Gap, orBestfit, which are programs in Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, Wis. FASTA provides alignments andpercent sequence identity of the regions of the best overlap between thequery and search sequences. See, e.g., Pearson, Methods Enzymol.183:63-98, 1990 (hereby incorporated by reference in its entirety). Forinstance, percent sequence identity between nucleic acid sequences canbe determined using FASTA with its default parameters (a word size of 6and the NOPAM factor for the scoring matrix) or using Gap with itsdefault parameters as provided in GCG Version 6.1, herein incorporatedby reference. Alternatively, sequences can be compared using thecomputer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410,1990; Gish and States, Nature Genet. 3:266-272, 1993; Madden et al.,Meth. Enzymol. 266:131-141, 1996; Altschul et al., Nucleic Acids Res.25:3389-3402, 1997; Zhang and Madden, Genome Res. 7:649-656, 1997,especially blastp or tblastn (Altschul et al., Nucleic Acids Res.25:3389-3402, 1997.

In some embodiments, the modified resilins differ from full-length ortruncated native resilins in amino acid residues that arepost-translationally modified (e.g., glycosylated, phosphorylated) suchthat the modified resilins have one or more different locations and/ordifferent amounts and/or different types of post-translationalmodifications than the full-length or truncated native resilins. In someembodiments, the modified resilins differ from full-length or truncatednative resilins in amino acid residues that are involved incross-linking such that the modified resilins have one or more differentlocations and/or different amounts and/or different types of amino acidsthat are involved in cross-linking than full-length or truncated nativeresilins. In some such embodiments, the modified resilins differ fromthe full-length or truncated native resilin in comprising one or moreadditional or fewer tyrosine residues, one or more additional or fewerlysine residues, and/or one or more additional or fewer cysteineresidues.

In some embodiments, the recombinant resilins comprise concatenatednative or truncated native resilins or concatenated modified resilins.In some embodiments, the concatenated native or truncated nativeresilins or concatenated modified resilins comprise at least 2 A-repeats(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, theconcatenated truncated native resilins or concatenated modified resilinscomprise at least 2 B-repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore).

The compositions provided herein comprise at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%; between 10% and 100%, 90%, 80%, 70%, 60%, 50%,40%, 30%, or 20%; between 20% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, or30%; between 30% and 100%, 90%, 80%, 70%, 60%, 50%, or 40%; between 40%and 100%, 90%, 80%, 70%, 60%, or 50%; between 50% and 100%, 90%, 80%,70%, or 60%; between 60% and 100%, 90%, 80%, or 70%; between 70% and100%, 90%, or 80%; between 80% and 100%, or 90%; or between 90% and 100%by weight of recombinant resilins. The recombinant resilins can beidentical recombinant resilins or mixtures of recombinant resilinshaving at least 2 different amino acid sequences.

In some embodiments, the compositions provided herein have similarproperties compared to compositions comprising native resilins. In otherembodiments, the compositions provided herein have different propertiescompared to compositions comprising native resilins. Non-limitedexamples of such properties include resilience, compressive elasticmodulus, tensile elastic modulus, shear modulus, extension to break,maximum tensile strength, hardness, rebound, and compression set.Parameters that can be modified to obtain compositions with specificmechanical properties include, for example, the length and/or sequenceof the recombinant resilins, the extent and/or type ofpost-translational modifications of the recombinant resilins, and/or theextent and/or type of cross-linking of the recombinant resilins.

In some embodiments, mechanical properties such as maximum tensilestrength, compressive elastic modulus, tensile elastic modulus, shearmodulus, extension to break and resilience can be measured using manydifferent types of tensile and compression systems that conductstress-strain measurements on elastomeric samples. The resultingstress-strain curves, including curves with hysteresis, can be measuredin tension or compression. In some embodiments, tension and compressiontest systems can apply a strain to a sample and measure the resultingforce using a load cell. In some embodiments, the mechanical propertiescan be measured at the macroscopic scale (e.g., using macroscopiccompression testers), microscopic, or nanoscopic scale (e.g., usingatomic-force microscopy (AFM) or nanoindentation measurements). In someembodiments, the compressive mechanical properties of elastomers can bemeasured according to the standard ASTM D575-91(2012) Standard TestMethods for Rubber Properties in Compression. Mechanical measurements ofelastomers in tension can be performed using ASTM D412-15a Standard TestMethods for Vulcanized Rubber and Thermoplastic Elastomers—Tension. Insome embodiments, tear strength of elastomers can be performed usingASTM D624-00 Standard Test Method for Tear Strength of ConventionalVulcanized Rubber and Thermoplastic Elastomers. In some embodiments,mechanical properties of slab, bonded, and molded elastomers can beperformed using ASTM D3574-11 Standard Test Methods for FlexibleCellular Materials—Slab, Bonded, and Molded Urethane Foams. In someembodiments, the mechanical properties of elastomers can be measuredusing ASTM D5992-96(2011) Standard Guide for Dynamic Testing ofVulcanized Rubber and Rubber-Like Materials Using Vibratory Methods.

In some embodiments, the compositions provided herein have a resilienceof greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, or greater than 95%; from 50% to 100%, 90%, 80%,70%, or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%,or 80%; from 80% to 100%, or 90%; from 90% to 100%; from 95% to 100%,from 90% to 99%, or from 95% to 99%.

In some embodiments, the compositions provided herein have a compressiveelastic modulus of less than 10 MPa, less than 7 MPa, less than 5 MPa,less than 2 MPa, less than 1 MPa, less than 0.5 MPa, or less than 0.1MPa; from 0.01 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, 0.5 MPa, or0.1 MPa; from 0.1 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, or 0.5 MPa;from 0.5 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, or 1 MPa; from 1 MPa to 10MPa, 7 MPa, 5 MPa, or 2 MPa; from 2 MPa to 10 MPa, 7 MPa, or 5 MPa; from5 MPa to 10 MPa, or 7 MPa; or from 7 MPa to 10 MPa. In some embodiments,the compressive elastic modulus of a composition can be measured asdefined by the ASTM D575-91(2012) Standard Test Methods for RubberProperties in Compression.

In some embodiments, the compositions provided herein have a tensileelastic modulus of less than 10 MPa, less than 7 MPa, less than 5 MPa,less than 2 MPa, less than 1 MPa, less than 0.5 MPa, or less than 0.1MPa; from 0.01 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, 1 MPa, or 0.5 MPa;from 0.5 MPa to 10 MPa, 7 MPa, 5 MPa, 2 MPa, or 1 MPa; from 1 MPa to 10MPa, 7 MPa, 5 MPa, or 2 MPa; from 2 MPa to 10 MPa, 7 MPa, or 5 MPa; from5 MPa to 10 MPa, or 7 MPa; or from 7 MPa to 10 MPa.

In some embodiments, the compositions provided herein have a shearmodulus of less than 1 MPa, less than 100 kPa, less than 50 kPa, lessthan 20 kPa, less than 10 kPa, or less than 1 kPa; from 0.1 kPa to 1MPa, 100 kPa, 50 kPa, 20 kPa, 10 kPa, or 1 kPa; from 1 kPa to 1 MPa, 100kPa, 50 kPa, 20 kPa, or 10 kPa; from 10 kPa to 1 MPa, 100 kPa, 50 kPa,or 20 kPa; from 20 kPa to 1 MPa, 100 kPa, or 50 kPa; from 50 kPa to 1MPa, or 100 kPa; or from 100 kPa to 1 MPa.

In some embodiments, the compositions provided herein have an extensionto break of greater than 1%, greater than 10%, greater than 50%, greaterthan 100%, greater than 300%, or greater than 500%; from 1% to 500%,300%, 100%, 50%, or 10%; from 10% to 500%, 300%, 100%, or 50%; from 50%to 500%, 300%, or 100%; from 100% to 500%, or 300%; or from 300% to500%.

In some embodiments, the compositions provided herein have a maximumtensile strength of greater than 0.1 kPa, greater than 1 kPa, greaterthan 2 kPa, greater than 5 kPa, or greater than 10 kPa; from 0.1 kPa to100 kPa, 10 kPa, 5 kPa, 2 kPa, or 1 kPa; from 1 kPa to 100 kPa, 10 kPa,5 kPa, or 2 kPa; from 2 kPa to 100 kPa, 10 kPa, or 5 kPa; from 5 kPa to100 kPa, or 10 kPa; or from 10 kPa to 100 kPa.

In some embodiments, mechanical properties such as hardness andcompressive elastic modulus can be measured using indentation andnanoindentation measurement systems. In some embodiments, indentationmeasurements utilizing a tip to indent the sample to a given amount ofstrain are used to measure the hardness and compressive elastic modulusof resilin, and the resulting force is measured using a load cell. Insome embodiments, different tip shapes can be used including Vickers andBerkovich shaped tips. In some embodiments, the hardness measured byindentation techniques is characterized by the relation, Hardness=(PeakForce)/(Contact Area).

In some embodiments, the hardness in polymers, elastomers, and rubberscan be measured using a durometer. In some embodiments the hardness ofan elastomer can be measured using the standard ASTM D2240, whichrecognizes twelve different durometer scales using combinations ofspecific spring forces and indentor configurations. The most commonscales are the Shore 00, A and D Hardness Scales. Hardness scales rangefrom 0 to 100, where 0 is softer material and 100 is harder material.

In some embodiments, the compositions provided herein have a Shore 00Hardness of less than 90, less than 80, less than 70, less than 60, lessthan 50, less than 40, less than 30, or less than 20; from 10 to 90, 80,70, 60, 50, 40, 30, or 20; from 20 to 90, 80, 70, 60, 50, 40, or 30;from 30 to 90, 80, 70, 60, 50, or 40; from 40 to 90, 80, 70, 60, or 50;from 50 to 90, 80, 70, or 60; from 60 to 90, 80, or 70; from 70 to 90,or 80; or from 80 to 90. In some embodiments, hardness measurements inresilin are performed according to ASTM D2240.

As used here, the term “rebound” refers to a particular measure ofresilience. In some embodiments, rebound can be measured with a numberof different tools including pendulum tools and dropped balls. In thependulum type measurements, RB, commonly called percentage rebound, isdetermined from the equation:

${RB} = {\frac{\left\lbrack {1 - {\cos\left( {{angle}\mspace{14mu}{of}\mspace{14mu}{rebound}} \right)}} \right\rbrack}{\left\lbrack {1 - {\cos\left( {{originial}\mspace{14mu}{angle}} \right)}} \right\rbrack} \times 100}$

The rebound resilience can be calculated as:

$R = \frac{h}{H}$

where h=apex height of the rebound, and H=initial height. The reboundresilience can also be determined by the measurement of the angle ofrebound. Some examples of test methods for determining rebound inelastomers are ASTM D2632-15 and ASTM D7121-05(2012).

In some embodiments, the compositions provided herein have a reboundgreater than 50%, greater than 60%, greater than 70%, greater than 80%,greater than 90%, or greater than 95%; from 50% to 100%, 90%, 80%, 70%,or 60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or80%; from 80% to 100%, or 90%; from 90% to 100%; from 95% to 100%, from90% to 99%, or from 95% to 99%. In some embodiments, reboundmeasurements in resilin are performed according to ASTM D2632-15, orASTM D7121-05(2012).

As used herein, the term “compression set” refers to a measure of thepermanent deformation remaining after an applied force is removed. Insome embodiments, compression set can be measured in different ways,including compression set under constant force in air (referred to asCompression Set A), compression set under constant deflection in air(referred to as Compression Set B), and compression set under constantdeflection in air considering material hardness (referred to asCompression Set C). Compression Set A (CA) is calculated by thefollowing expression: CA=[(t_(o)−t_(i))/t_(o)]×100, where t_(o) is theoriginal specimen thickness, and t_(i) is the specimen thickness aftertesting). Compression set B (C_(B)) is given byC_(B)=[(t_(o)−t_(i))/(t_(o)−t_(n))]100, where t_(o) is the originalspecimen thickness, t_(i) is the specimen thickness after testing, andt_(o) is the spacer thickness or the specimen thickness during the test.Some examples of test methods for determining compression set inelastomers are ASTM D3574-11 and ASTM D395-16.

In some embodiments, the compositions provided herein have a CompressionSet A or a Compression Set B of greater than 50%, greater than 60%,greater than 70%, greater than 80%, greater than 90%, or greater than95%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%, 90%,80%, or 70%; from 70% to 100%, 90%, or 80%; from 80% to 100%, or 90%;from 90% to 100%; from 95% to 100%, from 90% to 99%, or from 95% to 99%.In some embodiments, compression set measurements in resilin areperformed according to ASTM D3574-11 and ASTM D395-16.

The processing and forming of resilin into products can take many formsfor different applications. Accordingly, the compositions providedherein can have any shape and form, including but not limited to gels,porous sponges, films, machinable solids, cast forms, molded forms, andcomposites.

The compositions provided herein have a number of uses, including butnot limited to applications in aerospace, automotive, sportingequipment, vibration isolation, footwear, and clothing among others.Some applications from these categories are listed as non-limitingexamples. Due to the desirable elastic efficiency, resilin can be usedas an energy storage device (e.g., a rubber band) for storing andrecovering mechanical energy. Automobile suspension systems can beimproved by application of resilin bushings to keep more tire contact onthe road when going over bumps and through potholes at speed.Additionally, there are a number of sporting equipment applications forresilin with differently tuned mechanical properties including cores ofgolf balls, tennis racket grips, golf club grips, and table tennispaddles.

An application of particular interest is footwear due to the uniqueproperties of resilin compositions provided herein. As an insole ormidsole, resilin can improve the comfort and bioefficiency of shoes bycushioning the foot strike and restoring more of the energy from thatfootstrike as forward momentum. As a midsole, resilin can make up theentire midsole or be encapsulated within another material to complementits properties (e.g., an abrasion or wear resistant material, or amaterial tuned for traction). The resilin midsole can also contain aplurality of resilin materials with differently tuned mechanicalproperties that work in concert to provide enhanced performance (e.g.,softer heel strike area and firmer arch support).

As used herein, the term “density” refers to the mass of the sampledivided by the volume. In some embodiments, the density of an elastomercan be determined using a pycnometer with alcohol in place of water toeliminate air bubbles. In some embodiments, the density of an elastomercan be determined using a hydrostatic method. As used herein, the term“compressed volume density” refers to the ratio of the sample mass tothe compressed volume of the sample, where the “compressed volume” isdefined as the final equilibrium volume attained by an elastomericsample when it is subjected to a compressive force sufficient to causeit to flow until it fully conforms to the surrounding shape of thepiston-cylinder test chamber enclosure. In some embodiments, thecompressed volume density of an elastomer can be determined using acompressed volume densimeter.

In some embodiments, the compositions provided herein have a density ora compressed volume density are from 0.5 mg/cm³ to 2.0 mg/cm³, or from1.0 mg/cm³ to 1.5 mg/cm³, or from 1.1 mg/cm³ to 1.4 mg/cm³, or from 1.2mg/cm³ to 1.35 mg/cm³. In some embodiments, he determination of thedensity or the compressed volume density of elastomers can be performedusing ASTM D297-15 Standard Test Methods for Rubber Products—ChemicalAnalysis.

Recombinant Resilin Vectors, Recombinantn Host Cells, and Fermentations

Further provided herein are vectors encoding recombinant resilins,recombinant host cells comprising such vectors, and fermentationscomprising such recombinant host cells and recombinant resilins.

In some embodiments, the vectors provided herein comprise secretedresilin coding sequences, which encode a resilin polypeptide fused atits N-terminus to a secretion signal and optionally at its C-terminus toa tag peptide or polypeptide. In some embodiments, the vectors comprisesecreted resilin coding sequences that are codon-optimized forexpression in a particular host cell.

Suitable secretion signals are secretion signals that mediate secretionof polypeptides in the recombinant host cells provided herein.Non-limiting examples of suitable secretion signals are the secretionsignals of the alpha mating factor (α-MF) of Saccharomyces cerevisiae,acid phosphatase (PHO1) of Pichia pastoris, and phytohemagglutinin(PHA-E) from the common bean Phaseolus vulgaris. Additional secretionsignals are known in the art, or can be identified by identification ofproteins secreted by a host cell followed by genomic analysis of thesecreted proteins and identification of the non-translated N-terminalsequences (see, for example, Huang et al. A proteomic analysis of thePichia pastoris secretome in methanol-induced cultures. Appl MicrobiolBiotechnol. 2011 April; 90(1):235-47).

The resilins encoded by the secreted resilin coding sequences can befurther fused to tag peptides or polypeptides. Non-limiting examples oftag peptides or polypeptides include affinity tags (i.e., peptides orpolypeptides that bind to certain agents or matrices), solubilizationtags (i.e., peptides or polypeptides that assist in proper folding ofproteins and prevent precipitation), chromatography tags (i.e., peptidesor polypeptides that alter the chromatographic properties of a proteinto afford different resolution across a particular separationtechniques), epitope tags (i.e., peptides or polypeptides that are boundby antibodies), fluorescence tags (i.e., peptides or polypeptides thatupon excitation with short-wavelength light emit high-wavelength light),chromogenic tags (i.e., peptides or polypeptides that absorb specificsegments of the visible light spectrum), enzyme substrate tags (i.e.,peptides or polypeptides that are the substrates for specific enzymaticreactions), chemical substrate tags (i.e., peptides or polypeptides thatare the substrates for specific chemical modifications), or combinationsthereof. Non-limiting examples of suitable affinity tags include maltosebinding protein (MBP), glutathione-S-transferase (GST), poly(His) tag,SBP-tag, Strep-tag, and calmodulin-tag. Non-limiting examples ofsuitable solubility tags include thioredoxin (TRX), poly(NANP), MBP, andGST. Non-limiting examples of chromatography tags include polyanionicamino acids (e.g., FLAG-tag [GDYKDDDDKDYKDDDDKDYKDDDDK (SEQ ID NO: 45)])and polyglutamate tag. Non-limiting examples of epitope tags includeV5-tag, VSV-tag, Myc-tag, HA-tag, E-tag, NE-tag, and FLAG-tag.Non-limiting examples of fluorescence tags include green fluorescentprotein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein(CFP), yellow fluorescent protein (YFP), orange fluorescent protein(OFP), red fluorescent protein (RFP), and derivatives thereof.Non-limiting examples of chromogenic tags include non-fluorescentmembers of the GFP-like family of proteins (e.g., BlitzenBlue,DonnerMagenta; DNA2.0, Neward, Calif.). Non-limiting examples of enzymesubstrate tags include peptides or polypeptides comprising a lysinewithin a sequence suitable for biotinilation (e.g., AviTag, BiotinCarboxyl Carrier Protein [BCCP]). Non-limiting examples of chemicalsubstrate tags include substrates suitable for reaction with FIAsH-EDT2.The fusion of the C-terminal peptide or polypeptide to the resilin canbe cleavable (e.g., by TEV protease, thrombin, factor Xa, orenteropeptidase) on non-cleavable.

In some embodiments, the vectors comprise single secreted resilin codingsequences. In other embodiments, the vectors comprise 2 or more (e.g.,3, 4, or 5) secreted resilin coding sequences. In some such embodiments,the secreted resilin coding sequences are identical. In other suchembodiments, at least 2 of the secreted resilin coding sequences are notidentical. In embodiments in which at least 2 of the secreted resilincoding sequences are not identical, the at least 2 secreted resilincoding sequences can differ from each other in the resilins and/or inthe secretion signals and/or the optional tag peptides or polypeptidesthey encode.

In some embodiments, the vectors comprise promoters that are operablylinked to the secreted resilin coding sequences such that they drive theexpression of the secreted resilin coding sequences. The promotors canbe constitutive promoters or inducible promoters. In some embodiments,induction of the inducible promoter occurs via glucose repression,galactose induction, sucrose induction, phosphate repression, thiaminerepression, or methanol induction. Suitable promoters include promotersthat mediate expression of proteins in the recombinant host cellsprovided herein. Non-limiting examples of suitable promoters include theAOX1 promoter, GAP promoter, LAC4-PBI promoter, T7 promoter, TACpromoter, GCW14 promoter, GAL1 promoter, XPL promoter, XPR promoter,beta-lactamase promoter, spa promoter, CYC1 promoter, TDH3 promoter, GPDpromoter, TEF1 promoter, ENO2 promoter, PGL1 promoter, SUC2 promoter,ADH1 promoter, ADH2 promoter, HXT7 promoter, PHOS promoter, and CLB1promoter. Additional promoters that can be used to facilitate expressionof the secreted resilin coding sequences are known in the art.

In some embodiments, the vectors comprise terminators that are operablylinked to the secreted resilin coding sequences such that they effecttermination of transcription of the secreted resilin coding sequences.Suitable terminators include terminators that terminate transcription inthe recombinant host cells provided herein. Non-limiting examples ofsuitable terminators include the AOX₁ terminator, PGK1 terminator, andTPS1 terminator. Additional terminators that effect termination oftranscription of the secreted resilin coding sequences are known in theart.

In embodiments in which the vectors comprise 2 or more resilin codingsequences, the 2 or more resilin coding sequences can be operably linkedto the same promoters and/or terminators or to 2 or more differentpromoters and/or terminators.

The vectors provided herein can further comprise elements suitable forpropagation of the vectors in recombinant host cells. Non-limitingexamples of such elements include bacterial origins of replication andselection markers (e.g., antibiotic resistance genes, auxotrophicmarkers). Bacterial origins of replication and selection markers areknown in the art. In some embodiments, the selection marker is a drugresistant marker. A drug resistant maker enables cells to detoxify anexogenously added drug that would otherwise kill the cell. Illustrativeexamples of drug resistant markers include but are not limited to thosefor resistance to antibiotics such as ampicillin, tetracycline,kanamycin, bleomycin, streptomycin, hygromycin, neomycin, Zeocin™, andthe like. In some embodiments, the selection marker is an auxotrophicmarker. An auxotrophic marker allows cells to synthesize an essentialcomponent (usually an amino acid) while grown in media that lacks thatessential component. Selectable auxotrophic gene sequences include, forexample, hisD, which allows growth in histidine-free media in thepresence of histidinol. Other selection markers suitable for the vectorsof the present invention include a bleomycin-resistance gene, ametallothionein gene, a hygromycin B-phosphotransferase gene, the AURIgene, an adenosine deaminase gene, an aminoglycoside phosphotransferasegene, a dihydrofolate reductase gene, a thymidine kinase gene, and axanthine-guanine phosphoribosyltransferase gene.

The vectors of the present invention can further comprise targetingsequences that direct integration of the secreted resilin codingsequences to specific locations in the genome of host cells.Non-limiting examples of such targeting sequences include nucleotidesequences that are identical to nucleotide sequences present in thegenome of a host cell. In some embodiments, the targeting sequences areidentical to repetitive elements in the genome of host cells. In someembodiments, the targeting sequences are identical to transposableelements in the genome of host cells.

In some embodiments, recombinant host cells are provided herein thatcomprise the vectors described herein. In some embodiments, the vectorsare stably integrated within the genome (e.g., a chromosome) of therecombinant host cells, e.g., via homologous recombination or targetedintegration. Non-limiting examples of suitable sites for genomicintegration include the Tyl loci in the Saccharomyces cerevisiae genome,the rDNA and HSP82 loci in the Pichia pastoris genome, and transposableelements that have copies scattered throughout the genome of therecombinant host cells. In other embodiments, the vectors are not stablyintegrated within the genome of the recombinant host cells but ratherare extrachromosomal.

Recombinant host cells can be of mammalian, plant, algae, fungi, ormicrobe origin. Non-limiting examples of suitable fungi includemethylotrophic yeast, filamentous yeast, Arxula adeninivorans,Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae,Candida etchellsii, Candida guilliermondii, Candida humilis, Candidalipolytica, Candida pseudotropicalis, Candida utilis, Candidaversatilis, Debaryomyces hansenii, Endothia parasitica, Eremotheciumashbyii, Fusarium moniliforme, Hansenula polymorpha, Kluyveromyceslactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans,Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor mieheivar. Cooney et Emerson, Mucor pusillus Lindt, Penicillium roquefortii,Pichia methanolica, Pichia pastoris (Komagataella phaffii), Pichia(Scheffersomyces) stipitis, Rhizopus niveus, Rhodotorula sp.,Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae,Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomycesellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus,Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe,Saccharomyces sake, Saccharomyces uvarum, Sporidiobolus johnsonii,Sporidiobolus salmonicolor, Sporobolomyces roseus, Trichoderma reesi,Xanthophyllomyces dendrorhous, Yarrowia lipolytica, Zygosaccharomycesrouxii, and derivatives and crosses thereof.

Non-limiting examples of suitable microbes include Acetobactersuboxydans, Acetobacter xylinum, Actinoplane missouriensis, Arthrospiraplatensis, Arthrospira maxima, Bacillus cereus, Bacillus coagulans,Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis,Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus,Lactobacillus reuteri, Lactococcus lactis, Lactococcus lactis LancefieldGroup N, Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostocmesenteroides strain NRRL B-512(F), Micrococcus lysodeikticus,Spirulina, Streptococcus cremoris, Streptococcus lactis, Streptococcuslactis subspecies diacetylactis, Streptococcus thermophilus,Streptomyces chattanoogensis, Streptomyces griseus, Streptomycesnatalensis, Streptomyces olivaceus, Streptomyces olivochromogenes,Streptomyces rubiginosus, Xanthomonas campestris, and derivatives andcrosses thereof. Additional strains that can be used as recombinant hostcells are known in the art. It should be understood that the term“recombinant host cell” is intended to refer not only to the particularsubject cell but to the progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but is still included within the scope of the term“recombinant host cell” as used herein.

In some embodiments, the recombinant host cells comprise geneticmodifications that improve production of the recombinant resilinsprovided herein. Non-limiting examples of such genetic modificationsinclude altered promoters, altered kinase activities, altered proteinfolding activities, altered protein secretion activities, altered geneexpression induction pathways, and altered protease activities.

The recombinant host cells provided herein are generated by transformingcells of suitable origin with vectors provided herein. For suchtransformation, the vectors can be circularized or be linear.Recombinant host cell transformants comprising the vectors can bereadily identified, e.g., by virtue of expressing drug resistance orauxotrophic markers encoded by the vectors that permit selection for oragainst growth of cells, or by other means (e.g., detection of lightemitting peptide comprised in vectors, molecular analysis of individualrecombinant host cell colonies, e.g., by restriction enzyme mapping, PCRamplification, or sequence analysis of isolated extrachromosomal vectorsor chromosomal integration sites).

In some embodiments, the recombinant host cells provided herein canproduce high titers of the recombinant resilins provided herein. In somesuch embodiments, the recombinant host cells produce the recombinantresilins at a rate of greater than 2 mg resilin/g dry cell weight/hour,4 mg resilin/g dry cell weight/hour, 6 mg resilin/g dry cellweight/hour, 8 mg resilin/g dry cell weight/hour, 10 mg resilin/g drycell weight/hour, 12 mg resilin/g dry cell weight/hour, 14 mg resilin/gdry cell weight/hour, 16 mg resilin/g dry cell weight/hour, 18 mgresilin/g dry cell weight/hour, 20 mg resilin/g dry cell weight/hour, 25mg resilin/g dry cell weight/hour, or 30 mg resilin/g dry cellweight/hour; from 2 to 40, 30, 20, 10, or 5 mg resilin/g dry cellweight/hour; from 5 to 40, 30, 20, or 10 mg resilin/g dry cellweight/hour; from 10 to 40, 30, or 20 mg resilin/g dry cell weight/hour;from 20 to 40, or 30 mg resilin/g dry cell weight/hour; or from 30 to 40mg resilin/g dry cell weight/hour. In other such embodiments, therecombinant host cells secrete the recombinant resilins at a rate ofgreater than 2 mg resilin/g dry cell weight/hour, 4 mg resilin/g drycell weight/hour, 6 mg resilin/g dry cell weight/hour, 8 mg resilin/gdry cell weight/hour, 10 mg resilin/g dry cell weight/hour, 12 mgresilin/g dry cell weight/hour, 14 mg resilin/g dry cell weight/hour, 16mg resilin/g dry cell weight/hour, 18 mg resilin/g dry cell weight/hour,20 mg resilin/g dry cell weight/hour, 25 mg resilin/g dry cellweight/hour, or 30 mg resilin/g dry cell weight/hour; from 2 to 40, 30,20, 10, or 5 mg resilin/g dry cell weight/hour; from 5 to 40, 30, 20, or10 mg resilin/g dry cell weight/hour; from 10 to 40, 30, or 20 mgresilin/g dry cell weight/hour; from 20 to 40, or 30 mg resilin/g drycell weight/hour; or from 30 to 40 mg resilin/g dry cell weight/hour.The identities of the recombinant resilins produced can be confirmed byHPLC quantification, Western blot analysis, polyacrylamide gelelectrophoresis, and 2-dimensional mass spectroscopy (2D-MS/MS) sequenceidentification.

In some embodiments, the recombinant host cells provided herein havehigh secreted fractions of the recombinant resilins provided herein. Insome such embodiments, the recombinant host cells have secretedfractions of recombinant resilient that is greater than 50%, 60%, 70%,80%, or 90%; from 50% to 100%, 90%, 80%, 70%, or 60%; from 60% to 100%,90%, 80%, or 70%; from 70% to 100%, 90%, or 80%; from 90% to 100%, or90%; or from 90% to 100%.

Production and secretion of recombinant resilins can be influenced bythe number of copies of the secreted resilin coding sequences comprisedin the recombinant host cells and/or the rate of transcription of thesecreted resilin coding sequences comprised in the recombinant hostcells. In some embodiments, the recombinant host cells comprise a singlesecreted resilin coding sequence. In other embodiments, the recombinanthost cells comprise 2 or more (e.g., 3, 4, 5, or more) secreted resilincoding sequences. In some embodiments, the recombinant host cellscomprise secreted resilin coding sequences that are operably linked tostrong promoters. Non-limiting examples of strong promoters include thepGCW14 promoter of Pichia pastoris. In some embodiments, the recombinanthost cells comprise secreted resilin coding sequences that are operablylinked to medium promoters. Non-limiting examples of such mediumpromoters include the pGAP promoter of Pichia pastoris. In someembodiments, the recombinant host cells comprise coding sequencesencoding resilins under the control of weak promoters.

The fermentations provided herein comprise recombinant host cellsdescribed herein and a culture medium suitable for growing therecombinant host cells.

The fermentations are obtained by culturing the recombinant host cellsin culture media that provide nutrients needed by the recombinant hostcells for cell survival and/or growth, and for secretion of therecombinant resilins. Such culture media typically contain an excesscarbon source. Non-limiting examples of suitable carbon sources includemonosaccharides, disaccharides, polysaccharides, and combinationsthereof. Non-limiting examples of suitable monosaccharides includeglucose, galactose, mannose, fructose, ribose, xylose, arabinose,ribose, and combinations thereof. Non-limiting examples of suitabledisaccharides include sucrose, lactose, maltose, trehalose, cellobiose,and combinations thereof. Non-limiting examples of suitablepolysaccharides include raffinose, starch, glycogen, glycan, cellulose,chitin, and combinations thereof.

In some embodiments, the fermentations comprise recombinant resilins inamounts of at least 1%, 5%, 10%, 20%, or 30%; from 1% to 100%, 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, or 10%; from 10% to 100%, 90%, 80%, 70%,60%, 50%, 40%, 30%, or 20%; from 20% to 100%, 90%, 80%, 70%, 60%, 50%,40%, or 30%; from 30% to 100%, 90%, 80%, 70%, 60%, 50%, or 40%; from 40%to 100%, 90%, 80%, 70%, 60%, or 50%; from 50% to 100%, 90%, 80%, 70%, or60%; from 60% to 100%, 90%, 80%, or 70%; from 70% to 100%, 90%, or 80%;from 80% to 100%, or 90%; or from 90% to 100% by weight of the totalfermentation.

In some embodiments, the fermentations comprise recombinant resilin inan amount of at least 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, or30 g/L; from 2 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L,60 g/L, 50 g/L, 40 g/L, 30 g/L, 20 g/L, or 10 g/L; from 10 g/L to 300g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, 50 g/L, 40 g/L,30 g/L, or 20 g/L; from 20 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80g/L, 70 g/L, 60 g/L, 50 g/L, 40 g/L, or 30 g/L; from 30 g/L to 300 g/L,200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, 50 g/L, or 40 g/L;from 40 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60g/L, or 50 g/L; from 50 g/L to 300 g/L, 200 g/L, 100 g/L, 90 g/L, 80g/L, 70 g/L, or 60 g/L; from 60 g/L to 300 g/L, 200 g/L, 100 g/L, 90g/L, 80 g/L, or 70 g/L; from 70 g/L to 300 g/L, 200 g/L, 100 g/L, 90g/L, or 80 g/L; from 80 g/L to 300 g/L, 200 g/L, 100 g/L, or 90 g/L;from 90 g/L to 300 g/L, 200 g/L, or 100 g/L; from 100 g/L to 300 g/L, or200 g/L; or from 200 g/L to 300 g/L.

Methods

Further provided herein are methods for the production of therecombinant resilins described herein.

The methods are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification unless otherwise indicated. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates,1992, and Supplements to 2002); Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1990; Taylor and Drickamer, Introduction to Glycobiology,Oxford Univ. Press, 2003; Worthington Enzyme Manual, WorthingtonBiochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section AProteins, Vol I, CRC Press, 1976; Handbook of Biochemistry: Section AProteins, Vol II, CRC Press, 1976; Essentials of Glycobiology, ColdSpring Harbor Laboratory Press, 1999.

In some embodiments, a novel method is utilized to secrete resilinextracellularly from a host cell. In some embodiments, the methodcomprises constructing a vector comprising a secreted resilin codingsequence (step 1001 in FIG. 2), transforming the vector into a host cell(step 1002 in FIG. 2), and then culturing the recombinant host cells tosecrete resilin extracellularly (step 1003 in FIG. 2). In someembodiments, the method includes secreting the resilin extracellularlyat a rate greater than 2 mg resilin/g dry cell weight/hour, 4 mgresilin/g dry cell weight/hour, 6 mg resilin/g dry cell weight/hour, 8mg resilin/g dry cell weight/hour, 10 mg resilin/g dry cell weight/hour,12 mg resilin/g dry cell weight/hour, 14 mg resilin/g dry cellweight/hour, 16 mg resilin/g dry cell weight/hour, 18 mg resilin/g drycell weight/hour, 20 mg resilin/g dry cell weight/hour, 25 mg resilin/gdry cell weight/hour, or 30 mg resilin/g dry cell weight/hour; from 2 to40, 30, 20, 10, or 5 mg resilin/g dry cell weight/hour; from 5 to 40,30, 20, or 10 mg resilin/g dry cell weight/hour; from 10 to 40, 30, or20 mg resilin/g dry cell weight/hour; from 20 to 40, or 30 mg resilin/gdry cell weight/hour; or from 30 to 40 mg resilin/g dry cellweight/hour. In some embodiments, the secreted resilin is then purified(step 1004 in FIG. 2), and the purified resilin is cross-linked to forman elastomer (step 1005 in FIG. 2). In some embodiments, the methodsprovided herein comprise the step of transforming cells with vectorsprovided herein to obtain recombinant host cells provided herein (step1002 in FIG. 2). Methods for transforming cells with vectors arewell-known in the art. Non-limiting examples of such methods includecalcium phosphate transfection, dendrimer transfection, liposometransfection (e.g., cationic liposome transfection), cationic polymertransfection, electroporation, cell squeezing, sonoporation, opticaltransfection, protoplast fusion, impalefection, hyrodynamic delivery,gene gun, magnetofection, and viral transduction. One skilled in the artis able to select one or more suitable methods for transforming cellswith vectors provided herein based on the knowledge in the art thatcertain techniques for introducing vectors work better for certain typesof cells.

In some embodiments, the methods further comprise the step of culturingthe recombinant host cells provided herein in culture media underconditions suitable for obtaining the fermentations provided herein(step 1003 in FIG. 2). In some embodiments, the conditions and culturemedia are suitable to facilitate secretion of the recombinant proteinsfrom the recombinant host cells into the culture media. Suitable culturemedia for use in these methods are known in the art, as are suitableculture conditions. Exemplary details of culturing yeast host cells aredescribed in Idiris et al., Appl. Microbiol. Biotechnol. 86:403-417,2010; Zhang et al., Biotechnol. Bioprocess. Eng. 5:275-287, 2000; Zhu,Biotechnol. Adv. 30:1158-1170, 2012; Li et al., MAbs 2:466-477, 2010.

In some embodiments, the methods further comprise the step of purifyingsecreted recombinant resilins from the fermentations provided herein toobtain the recombinant resilins provided herein (step 1004 in FIG. 2).Purification can occur by a variety of methods known in the art forpurifying secreted proteins from fermentations. Common steps in suchmethods include centrifugation (to remove cells) followed byprecipitation of the proteins using precipitants or other suitablecosmotropes (e.g., ammonium sulfate). The precipitated protein can thenbe separated from the supernatant by centrifugation, and resuspended ina solvent (e.g., phosphate buffered saline [PBS]). The suspended proteincan be dialyzed to remove the dissolved salts. Additionally, thedialyzed protein can be heated to denature other proteins, and thedenatured proteins can be removed by centrifugation. Optionally, thepurified recombinant resilins can be coacervated.

In various embodiments, methods of purifying the secreted recombinantproteins from the fermentation can include various centrifugation stepsin conjunction with solubilizing protein in a whole cell broth or cellpellet with known chaotropes such as urea or guanidine thiocyanate.

In some embodiments, the methods provided herein further comprise thestep of cross-linking the recombinant resilins to obtain the recombinantresilin compositions provided herein (step 1005 in FIG. 2). Methods forcross-linking proteins are known in the art. In some embodiments,cross-linking is achieved via enzymatic cross-linking (e.g., usinghorseradish peroxidase). In other embodiments, cross-linking is achievedvia photochemical cross-linking (see, for example, Elvin C M, Carr A G,Huson M G, Maxwell J M, Pearson R D, Vuocolo T, Liyou N E, Wong D C C,Merritt D J, Dixon N E. Nature 2005, 437, 999-1002; Whittaker J L, DuttaN K, Elvin C M, Choudhury N R. Journal of Materials Chemistry B 2015, 3,6576-79; Degtyar E, Mlynarczyk B, Fratzl P, Harrington M J. Polymer2015, 69, 255-63). In some embodiments, cross-linking is achieved viachemical cross-linking (see, for example, Renner J N, Cherry K M, Su R SC, Liu J C. Biomacromolecules 2012, 13, 3678-85; Charanti, M B,Ifkovits, J L, Burdick, J A, Linhardt J G, Kiick, K L. Soft Matter 2009,5, 3412-16; Li L Q, Tong Z X, Jia X Q, Kiick K L. Soft Matter 2013, 9,665-73; Li L, Mahara A, Tong Z, Levenson E A, McGann C L, Jia X, YamaokaT, Kiick K L. Advanced Healthcare Materials 2016, 5, 266-75). In someembodiments, cross-linking is achieved via tyrosine residues. In otherembodiments, cross linking is achieved via lysine residues. In someembodiments, cross linking is achieved via cysteine residues. In someembodiments, cross-linking employs transglutaminase (see, for example,Kim Y, Gill E E, Liu J C. Enzymatic Cross-Linking of Resilin-BasedProteins for Vascular Tissue Engineering Applications.Biomacromolecules. 17(8):2530-9). In some embodiments, cross-linkingemploys poly(ethylene glycol) (PEG) (McGann C L, Levenson E A, Kiick KL. Macromol. Chem. Phys. 2013, 214, 203-13; McGann C L, Akins R E, KiickK L. Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffoldswith Heterogeneous Microstructure. Biomacromolecules. 2016;17(1):128-40). In some embodiments, cross-linking occurs in vessels ormolds such that the recombinant resilin compositions obtained havespecific shapes or forms.

EXAMPLES Example 1: Generation of Pichia pastoris Recombinant Host Cellsthat Secrete Recombinant Resilin

Pichia pastoris recombinant host cells that secrete recombinant resilinwere generated by transforming a HIS+ derivative of GS115 (NRRL Y15851)Pichia pastoris (Komagataella phaffii) with vectors comprising secretedresilin coding sequences.

The vectors each comprised 3 resilin coding sequences fused in frame toan N-terminal secretion signal (alpha mating factor leader and prosequence), and in some instances a C-terminal 3×FLAG tag (SEQ ID NO: 45)(see FIG. 3). Each of the secreted resilin coding sequences was flankedby a promoter (pGCW14) and a terminator (tAOX1 pA signal). The vectorsfurther comprised a targeting region that can direct integration of the3 secreted resilin coding sequences to the HSP82 locus of the Pichiapastoris genome, dominant resistance markers for selection of bacterialand yeast transformants, as well as a bacterial origin of replication.

The resilin coding sequences were obtained from scientific literatureand from searching public sequence databases. The nucleotide sequenceswere translated into amino acid sequences and then codon-optimized. Bothfull length and truncated resilin sequences were chosen. Selectedsecreted resilin coding sequences are listed in Table 1.

TABLE 1 Exemplary full-length and truncated resilin amino acid sequencesand recombinant host strains Amino Acid Short SEQ ID With FLAG tagWithout FLAG tag Species Type Name NO: Plasmid Strain Plasmid StrainDrosophila Full length Ds_ACB 1 RMp4830 RMs1209 RMp4842 RMs1221sechellia Drosophila A repeats + Ds_AC 2 RMp4831 RMs1210 RMp4843 RMs1222sechellia Chitin binding domain Drosophila A repeats only Ds_A 3 RMp4832RMs1211 RMp4844 RMs1223 sechellia Acromyrmex A repeats only Ae_A 4RMp4833 RMs1212 RMp4845 RMs1224 echinatior Aeshna sp. B repeats onlyAs_B 5 RMp4834 RMs1213 RMp4846 RMs1225 Aeshna sp. Full length As_ACB 6RMp4835 RMs1214 RMp4847 RMs1226 Haematobia A repeats only Hi_A 7 RMp4836RMs1215 RMp4848 RMs1227 irritans Haematobia Full length Hi_ACB 8 RMp4837RMs1216 RMp4849 RMs1228 irritans Ctenocephalides A repeats only Cf_A 9RMp4838 RMs1217 RMp4850 RMs1229 felis Ctenocephalides B repeats onlyCf_B 10 RMp4839 RMs1218 RMp4851 RMs1230 felis Bombus A repeats only Bt_A11 RMp4840 RMs1219 RMp4852 RMs1231 terrestris Tribolium A repeats onlyTc_A 12 RMp4841 RMs1220 RMp4853 RMs1232 castaneum

The vectors were transformed into Pichia pastoris using electroporationto generate host strains comprising 3 integrated copies of each secretedresilin coding sequence. Transformants were plated on YPD agar platessupplemented with an antibiotic, and incubated for 48 hours at 30° C.

Clones from each final transformation were inoculated into 400 μL ofBuffered Glycerol-complex Medium (BMGY) in 96-well blocks, and incubatedfor 24 hours at 30° C. with agitation at 1,000 rpm. A sample wasremoved, the recombinant host cells were pelleted via centrifugation,and the supernatant was recovered and run on a SDS-PAGE gel for analysisof resilin content via Coomassie gel and Western blot analysis (forpolypeptides comprising the 3×FLAG tag). For FLAG-tagged proteins, theremaining cultures were used to inoculate minimal media cultures induplicate for ELISA measurements. One duplicate was pelleted and thesupernatant was measured directly. The second duplicate was extractedwith guanidine thiocyanate and both the intra- and extra-cellularfractions were measured.

As shown in FIG. 4B and FIG. 4C, recombinant resilin from numerousspecies expressed successfully in the Pichia pastoris recombinant hostcells. (Note: Some proteins have very few basic residues, and aretherefore difficult to detect by Coomassie, though they have a signal onWestern.) As shown in FIG. 4A, recombinant host cells secreted up to 90%of the recombinant resilin produced.

Example 2: Measuring Productivity of Pichia pastoris Recombinant HostCells Expressing and Secreting Recombinant Resilin

To measure productivity, 3 clones of each recombinant host cell wereinoculated into 400 μL of BMGY in a 96-well square-well block, andincubated for 48 hours at 30° C. with agitation at 1,000 rpm. Followingthe 48-hour incubation, 4 μL of each culture was used to inoculate 400μL of minimal media in a 96-well square-well block, which was thenincubated for 48 hours 30° C. with agitation at 1,000 rpm. 400 uL of 5Mguanidine thiocyanate was added to the cultures, and the mixtures werepelleted by centrifugation. The supernatants were saved whereas thepellets were resuspended in 800 μL of 2.5M guanidine thiocyanate. Theresuspended cells were physically lysed using beads, the lysed cellmixture was pelleted by centrifugation, and the supernatant was saved.The concentration of resilin in each fraction was determined by directenzyme-linked immunosorbent assay (ELISA) analysis quantifying the3×FLAG epitope (FIG. 5A and FIG. 5B).

Example 3: Purification of Recombinant Resilin

The non-FLAG-tagged Ds_ACB and Ae_A polypeptides were chosen forpurification and cross-linking. Strains RMs1221 (expressing Ds_ACB) andRMs1224 (expressing Ae_A) were grown in 500 mL of BMGY in flasks for 48hours at 30° C. with agitation at 300 rpm.

The protocol for purification was adapted from Lyons et al. (2007).Cells were pelleted by centrifugation, and supernatants were collected.Proteins were precipitated by addition of ammonium sulfate. Theprecipitated proteins were resuspended in a small volume of phosphatebuffered saline (PBS), and the resuspended samples were dialyzed againstPBS to remove salts. The dialyzed samples were then heated to denaturenative proteins, and denatured proteins were removed by centrifugation.The retained supernatants contained the purified resilin polypeptides.Optionally, the retained supernatants were chilled, which causedcoacervation, resulting in a concentrated lower phase and dilute upperphase.

As shown in FIG. 6, Ae_A was obtained in relatively pure form whereasDs_ACB produced 3 bands at 70 kDa, 50 kDa, and 25 kDa.

Example 4: Cross-Linking of Purified, Secreted, Recombinant Resilin

Concentrated Ds_ACB resilin was cross-linked via one of two methods:photo cross-linking (adapted from Elvin et al. 2005) and enzymaticcross-linking (adapted from Qin et al. 2009).

For photo cross-linking, resilin protein was mixed with ammoniumpersulfate and tris (bipyridine) ruthenium (II) ([Ru(bpy)3]2+). Themixture was exposed to bright white light, after which the mixtureformed a rubbery solid.

For enzymatic cross-linking, resilin protein was mixed with horseradishperoxidase (HRP) and hydrogen peroxide. The mixture was incubated at 37°C., after which the mixture formed a rubbery solid.

Example 5: Production of a Block of Recombinant Resilin

Strain RMs1221 (expressing the Ds_ACB resilin) was run in two 2 Lfermentation tanks to produce a larger quantity of protein.

The strain was grown in a minimal basal salt media with 15 g/L ofglucose as a starting feedstock and 1 g/L L81 antifoam, in a stirredfermentation vessel controlled at 30° C., with 1 VVM of air flow andminimum agitation of 700 rpm. The pH of the fermentation was controlledat 5 with on-demand addition of ammonium hydroxide. Once batch glucosewas depleted, glucose was added via a programmed feed recipe that wasdesigned to maintain the oxygen uptake rate 120 mmole/L/h, thetemperature was decreased to 25° C., and dissolved oxygen was maintainedat 20%. The fermentation was harvested after 70 hours, at about 700-800OD of cell density.

The protein was purified as described in Example 3, and combined withreagents for enzymatic cross-linking as described in Example 4. Thecross-linking mixture was filled into small cylindrical, rectangular,spherical, and shoe-shaped molds, and finally incubated at 37° C.Resulting recombinant resilin solids are shown in FIG. 7.

Example 6: Material Testing of Resilin Solids

A resilin cylinder produced as described in Example 5 was subjected to acompression test using a rheometer. The recombinant resilin cylindercould be compressed from an initial height of 7.3 mm (avg width 5.4 mm)to less than 0.66 mm without any breakage. As shown in FIG. 8, thecylinder returned to a height of 6.7 mm (avg width 5.6 mm) upon releaseof the compressive load.

Example 7: Methods for Recovering Full-Length Recombinant Resilin fromWhole Cell Broth

Various recovery and separation techniques were used to purify Ds_ACB(SEQ ID NO: 1) that was produced in strains with a 3× FLAG tag (RMs1209)and without a 3× FLAG tag (RMs1221) according to Example 1 above.

A first set of samples was prepared by centrifuging a whole cell brothto produce a first pellet of cells and a first supernatant, andextracting the first supernatant to produce a clear cell broth. Thefirst supernatant was then precipitated using ammonium sulfate andcentrifuged to produce a second pellet and second supernatant which wasdiscarded. The second pellet was then re-suspended in PBS for dialysis.The dialyzed solution was then subject to high temperature to denatureproteins other than Ds_ACB, which is stable at high temperatures. Thedenatured proteins were removed by centrifuging the dialyzed anddenatured solution to produce a third pellet and third supernatant. Thethird supernatant was retained from the denatured solution, thencoacervated by chilling the third supernatant to induce a phaseseparation into a dense lower layer containing the Ds_ACB and an upperlayer. These samples are referred to in Table 2 below and elsewhereherein as the “CCB” samples. In some CCB samples, multiple coacervationswere performed by retaining the lower layer and incubating the lowerlayer at a lower temperature to induce further phase separation. TheseCCB samples are respectively referred to in Table 2 below and elsewhereherein as the “first coacervation” and “second coacervation” samples.

A second set of samples was prepared by centrifuging a whole cell brothto produce a first pellet of cells and protein proximal to the cells(e.g. adherent to the cells, on the surface of the cells) and/orinsoluble protein (e.g. protein aggregates) and first supernatant, thendiscarding the first supernatant to obtain the first pellet. The firstpellet was re-suspended in guanidine thiocyanate to solubilize Ds_ACB.The re-suspension was centrifuged again produce a second pellet and asecond supernatant. The second supernatant was then dialyzed against PBSand subject to high temperature in order to denature proteins other thanDs_ACB and centrifuged to produce a third pellet and third supernatant.The third supernatant was subject to coacervation by chilling to yieldphase separation into a dense lower layer containing Ds_ACB and an upperlayer. These samples are referred to in Table 2 below and elsewhereherein as the “gel layer” samples. In some gel layer samples, multiplecoacervations were performed by retaining the lower layer and incubatingthe lower layer at a lower temperature to induce further phaseseparation. These gel layer samples are referred to in Table 2 below andelsewhere herein as the “first coacervation” and “second coacervation”samples.

A third set of samples was prepared by centrifuging a whole cell brothto produce a pellet and supernatant, then discarding the supernatant toobtain a pellet of cells and protein proximal to the cells (e.g.adherent to the cells, on the surface of the cells) and/or insolubleprotein (e.g. protein aggregates). The pellet of cells was re-suspendedin guanidine thiocyanate to solubilize the protein that was proximal tothe cells. The re-suspension was centrifuged again produce a secondpellet of cells and a second supernatant. The second supernatant wasthen precipitated with ammonium sulfate and centrifuged to produce athird pellet and third supernatant. The third pellet was suspended inguanidine thiocyanate, then dialyzed against PBS and subject to hightemperature to denature proteins other than Ds_ACB and centrifuged toproduce a fourth supernatant and fourth pellet. The fourth supernatantwas then subject to coacervation by chilling to yield phase separation.These samples are referred to in Table 2 below and elsewhere herein asthe “gel layer precipitated” samples.

A single sample was produced by adding urea to a whole cell broth tosolubilize the protein, then centrifuging the whole cell broth toproduce a first pellet and first supernatant. The first supernatant wasthen precipitated using ammonium sulfate and centrifuged to produce asecond pellet and second supernatant. The second supernatant wasdiscarded and the second pellet was then re-suspended in guanidinethiocyanate and dialyzed against PBS, then subject to high temperaturein order to denature proteins other than Ds_ACB and centrifuged again toproduce a third pellet and a third supernatant. The third supernatantwas then coacervated by chilling the third supernatant to induce a phaseseparation into a dense lower layer containing Ds_ACB and an upperlayer. This sample is referred to in Table 2 below and elsewhere hereinas the “Urea WCBE” sample.

Another single sample was prepared by centrifuging a whole cell broth toproduce a first pellet and first supernatant, then discarding the firstsupernatant to obtain a first pellet of cells and protein proximal tothe cells (e.g. adherent to the cells, on the surface of the cells)and/or insoluble protein (e.g. protein aggregates). The first pellet ofcells was re-suspended in guanidine thiocyanate to solubilize theprotein. The re-suspension was centrifuged again to produce a secondpellet of cells and a second supernatant. The second supernatant wasthen dialyzed against PBS and then centrifuged to produce a heavy phaseof protein, a light phase of supernatant and a film separating the heavyphase from the light phase. The heavy phase of protein was then isolatedby discarding the light phase and the film. This sample is referred toin Table 2 below and elsewhere herein as the “Dense layer” sample.

Table 2 (below) lists the various combinations of strains and recoverytechniques along with the relative amount of degradation seen in the gelpictured at FIG. 9. As shown in FIG. 9, samples E, F, G, K and L showedbands at approximately 110 kDa and minimal or faint bands at lowermolecular weights (labeled in Table 2 as “Minimal”). Samples A, B, C, D,G, I and J had degradation products corresponding to bands atapproximately 90, 30, 22, 17 and 12 kDa (labelled in Table 2 as“Substantial”). Among these, samples A and I also showed bands atapproximately 110 kDa indicating the presence of full-length resilin.Accordingly, the “Gel layer” samples produced full-length resilin whilethe CCB samples produced degradation products sometimes in addition tofull-length resilin (e.g. sample A) or without full-length resilin (e.g.samples C and D). The Urea WCBE sample only produced degradationproducts. CCB/gel layer precipitated indicates the combination ofisolated material from both the CCB purification and the gel layerpurification methods.

TABLE 2 Samples from recovery methods yielding full-length resilin anddegradation products Sample Strain FLAG Description Coacervation 110 kDaband? Degradation A RMs1221 − CCB First Yes Substantial B RMs1221 − UreaWCBE None No Substantial C RMs1221 − CCB First No Substantial D RMs1221− CCB Second No Substantial E RMs1221 − Gel layer First Yes Minimal FRMs1221 − Gel layer Second Yes Minimal G RMs1209 + CCB First NoSubstantial H RMs1209 + Gel layer First Yes Minimal precipitated IRMs1209 + CCB/gel layer First Yes Substantial precipitated J RMs1221 −CCB First No Substantial K RMs1221 − Gel layer First Yes Minimal LRMs1221 − Dense layer None Yes Minimal

To verify that the 110 kDa bands shown in samples A, I, E, F, G, K and Lcorresponded to the full-length resilin (SEQ ID NO: 1), the 110 kDa bandin sample H (indicated in FIG. 9 with an arrow) was excised and sent forN-terminus sequencing by Edman degradation. Edman degradation is acyclic procedure where amino acid residues are cleaved off one at a timeand identified by chromatography. There are 3 steps in the cyclicprocedure. In step 1 the PITC reagent is coupled to the N-terminal aminogroup under alkaline conditions. In step 2 the N-terminal residue iscleaved in acidic media. In step 3, the PITC coupled residue istransferred to a flask, converted to a PTH-residue and identified byHPLC chromatography. The next cycle is then started for identificationof the next N-terminal residue. Edman degradation analysis was performedon a Shimadazu PPSQ-33 sequencer and a PVDF membrane.

FIG. 10 shows the full-length Drosophila sechellia resilin sequence(Ds_ACB) that is expressed along with signal sequences that are latercleaved. The first sequence (italics), is an alpha mating factorprecursor protein signal sequence (SEQ ID NO: 46) that is cleaved twiceafter transcription by a signal peptidase followed by cleavage withKex2. The second sequence (bold) is an EAEA repeat that is cleaved bySte13 (SEQ ID NO: 47). The third sequence (lower case) corresponds toDrosophila sechellia full-length resilin (SEQ ID NO 1). The fourthsequence (bold and italicized) corresponds to a linker sequence (SEQ IDNO: 46). The fifth sequence (underlined) corresponds to the 3× FLAG tag(SEQ ID NO: 45).

Edman sequencing confirmed that the N-terminus of the protein sequencesat the approximately 110 kDa band corresponded to the full-length lengthDrosophila sechellia resilin sequence. Specifically, the N-terminussequencing showed that the N-terminus either corresponded to “EAEA” or“GRPE”, respectively the full-length Drosophila sechellia resilinsequence with or without the EAEA repeat.

Example 8: Quantifying the Stability of Crosslinked Resilin

Resilin samples generated by the methods described in Example 7 withvarying levels of degradation products and full-length resilin weresubject to enzymatic cross-linking as described above with respect toExample 4. The stability of the cross-linked samples was assessed overtime by determining the duration each cross-linked samples remained asolid through daily observation. Table 3 shows the time as a solid foreach cross-linked sample. As shown in Table 3, samples comprisingfull-length resilin had a longer duration of stability than the samplesthat did not comprise full-length resilin.

TABLE 3 Stability of cross-linked resilin Sample 110 kDa band?Degradation Time as solid A Yes Substantial 13 days B No Substantial  8days C No Substantial  6 days D No Substantial  6 days E Yes Minimal N/AF Yes Minimal 27 days G No Substantial  8 days H Yes Minimal 15 days IYes Substantial 15 days J No Substantial  6 days K Yes Minimal 13 days LYes Minimal 13 days

ADDITIONAL CONSIDERATIONS

The foregoing description of the embodiments of the disclosure has beenpresented for the purpose of illustration; it is not intended to beexhaustive or to limit the claims to the precise forms disclosed.Persons skilled in the relevant art can appreciate that manymodifications and variations are possible in light of the abovedisclosure.

The language used in the specification has been principally selected forreadability and instructional purposes, and it may not have beenselected to delineate or circumscribe the inventive subject matter. Itis therefore intended that the scope of the disclosure be limited not bythis detailed description, but rather by any claims that issue on anapplication based hereon. Accordingly, the disclosure of the embodimentsis intended to be illustrative, but not limiting, of the scope of theinvention, which is set forth in the claims.

SEQUENCE LISTING

SEQ ID NO: Species Sequence 1 DrosophilaRPEPPVNSYLPPSDSYGAPGQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPG sechellia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rosophilaRPEPPVNSYLPPSDSYGAPGQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPG sechelliaQGQGQGQGQGGYGGKPSDSYGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGSAGRPSSSYGAPGSGPGGRPSDSYGPPASG 3 DrosophilaGKPSDSYGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPS sechelliaDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGA PGSGNGS 4 AcromyrmexFGENRGNGGKPSTSYGVPDSNGNNRGGFGNGGSEGRPSTSYGLPDASRNNG echinatiorNGFGNVGNEDKPSTNYGIPANGNKVSGFGNVGSEGRPSTSYGVPGANGNQGFGSGGIGGRPSTSYGVPGVNGNNGGGFENVGRPSTSYGTPDARGNNGGSFRNGDIGGRPSTNYGIPGANGNHG 5 AeshnaAPSRGGGHGGGSISSSYGAPSKGSGGFGGGSISSSYGAPSKGSVGGGVSSSYGAPAIGGGSFGGGSFGGGSFGGGSFGGGAPSSSYGAPSSSYSAPSSSYGAPSKGSGGFGSSGGFSSFSSAPSSSYGAPSASYSTPSSSYGAPSSGGFGAGG GFSSG 6 AeshnaEPPVGGSQSYLPPSSSYGAPSAGTGFGHGGGSPSQSYGAPSFGGGSVGGGSHFGGGSHSGGGGGGYPSQSYGAPSRPSGSSFQAFGGAPSSSYGAPSSQYGAPSGGGGSYAIQGGSFSSGGSRAPSQAYGAPSNNAGLSHQSQSFGGGLSSSYGAPSAGFGGQSHGGGYSQGGNGGGHGGSSGGGYSYQSFGGGNGGGHGGSRPSSSYGAPSSSYGAPSGGKGVSGGFVSQPSGSYGAPSQSYGAPSRGGGHGGGSISSSYGAPSKGSGGFGGGSISSSYGAPSKGSVGGGVSSSYGAPAIGGGSFGGGSFGGGSFGGGSFGGGAPSSSYGAPSSSYSAPSSSYGAPSKGSGGFGSSGGFSSFSSAPSSSYGAPSASYSTPSSSYGAPSSGGFGAGGGFSSGGYSGGGGGYSSGGSGGFGGHGGSGGAGGYSGGGGYSGGGSGGGQKYDSNGGYVYS 7 HaematobiaAGGGNGGGGTGGTPSSSYGAPSNGGGSNGNGFGSPSSSYGAPGSGGSNGNG irritansGGRPSLSYGAPGSGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGS RPSSTYGAP 8Haematobia RPEPPVNSYLPPPLNNYGAPGAGGGSSDGSPLAPSDAYGAPDLGGGSGGSG irritansQGPSSSYGAPGLGGGNGGAPSSSYGAPGLGGGNGGSRRPSSSYGAPGAGGGNGGGGTGGTPSSSYGAPSNGGGSNGNGFGSPSSSYGAPGSGGSNGNGGGRPSLSYGAPGSGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGGRPSSSYGAPGAGGSNGNGGSRPSSTYGAPGAGGSNGNGCGNKPSSSYGAPSAGSNGNGGSEQGSSGSPSDSYGPPASGTGRGRNGGGGGAGGGRRGQPNQEYLPPNQGDNGNNGGSGGDDGYDYSQSGDGGGQGGSGGSGNGGDDGSNIVEYEAGQEGYRPQIRYEGEANEGGQGSGGAGGSDGTDGYEYEQNGGDGGAGGSGGPGTGQDLGENGYSSGRPGGDNGGGGGYSNGNGQGDGGQDLGSNGYSSGAPNGQNGGRRNGGGQNNNGQGYSSGRPNGNGSGGRNGNGGRGNGGGYRNGNGNGGGNGNGSGSGSGNNGYNYDQQGSN GFGAGGQNGENDGSGYRYS9 Ctenocephalides ANGNGFEGASNGLSATYGAPNGGGFGGNGNGGAPSSSYGAPGAGNGGNGGGfelis RPSSSYGAPGAGGSGNGFGGRPSSSYGAPGNGNGANGGRGGRPSSRYGAPGNGNGNGNGNGGRPSSSYGAPGSNGNGGRPSSSYGAPGSGNGFGGNGGRPSSSYGAPGANGNGNGGAIGQPSSSYGAPGQNGNGGGLSSTYGAPGAGNGGFGGNGGGLSSTYGAPGSGNGGFGGNGLSSTYGAPGSGNGGFGGNGGGLSSTYGA P 10 CtenocephalidesPGGAGGAGGYPGGAGGAGGAGGYPGGSAGGAGGYPGGSGSGVGGYPGGSNG felisGAGGYPGGSNGGAGGYPGGSNGGAGGYPGGSNGGAGGYPGGSNGNGGYSNGGSNGGGAGGYPGGSNGNGGYPGSGSNGGAGGYPGGSNGNGGYPG 11 BombusFDGQNGIGGGDSGRNGLSNSYGVPGSNGGRNGNGRGNGFGGGQPSSSYGAP terrestrisSNGLGGNGGSGAGRPSSSYGAPGGGNGFGGGQPSSSYGAPSNGLGGNGAGRPSSSYGAPGGGNGFGGGSNGAGKNGFGGAPSNSYGPPENGNGFGGGNGGGSPSGLYGPPGRNGGNGGNGGNGGNGGRPSSSYGTPERNGGRPSGLYGPP 12 TriboliumNGFGGGQNGGRLSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGG castaneumGQNGGRPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQGGNGFGGGQNGGKPSSTYGPPGQGGNGFGGGQNGGRPSSTYGPPGQG 13 TriboliumRAEPPVNSYLPPSQNGGPSSTYGPPGFQPGTPLGGGGNGGHPPSQGGNGGF 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 14 TriboliumQLTKRDAPLSGGYPSGGPANSYLPPGGASQPSGNYGAPSGGFGGKSGGFGG castaneumSGGFGGAPSQSYGAPSGGFGGSSSFGKSGGFGGAPSQSYGAPSGGFGGSSSFGKSSGGFGGAPSQSYGAPSGGFGGSSSFGKSGGFGGAPSQSYGAPSGGFGGSSSFGKSGGFGGAPSQSYGAPSGGFGGKSSSFSSAPSQSYGAPSGGFGGKSGGFGGAPSQSYGAPSGGFGGKSGGFGGAPSQSYGAPSGGFGGSSSFGKSGGFGGAPSQSYGAPSGGFGGSSSFGKSSGFGHGSGAPSQSYGAPSRSQPQSNYLPPSTSYGTPVSSAKSSGSFGGAPSQSYGAPSQSHAPSQSYGAPSRSFSQAPSQSYGAPSQGHAPAPQQSYSAPSQSYGAPSGGFGGGHGGFGGQGQGFGGGRSQPSQSYGAPAPSQSYGAPSAGGQQYASNGGYSY 15 Apis melliferaRSEPPVNSYLPPSGNGNGGGGGGSSNVYGPPGFDGQNGIGEGDNGRNGISNSYGVPTGGNGYNGDSSGNGRPGTNGGRNGNGNGRGNGYGGGQPSNSYGPPSNGHGGNGAGRPSSSYGAPGGGNGFAGGSNGKNGFGGGPSSSYGPPENGNGFNGGNGGPSGLYGPPGRNGGNGGNGGNGGRPSGSYGTPERNGGRLGGLYGAPGRNGNNGGNGYPSGGLNGGNGGYPSGGPGNGGANGGYPSGGSNGDNGGYPSGGPNGNGNGNGGYGQDENNEPAKYEFSYEVKDEQSGADYGHTESRDGDRAQGEFNVLLPDGRKQIVEYEADQDGFKPQIRYEGEANSQGYGSGGPGGNGGDNGYPSGGPGGNGYSSGRPNGGSDFSDGGYPSTRPGGENGGYRNGNNGGNGNG GYPSGNGGDAAANGGYQY16 Apis mellifera DAPISGSYLPPSTSYGTPNLGGGGPSSTYGAPSGGGGGRPSSSYGAPSSTYGAPSSTYGAPSNGGGRPSSTYGAPSNGGGRPSSSYGAPSSSYGAPSSTYGAPSNGGGRPSSSYGAPSFGGGGGFGGGNGLSTSYGAPSRGGGGGGGSISSSYGAPTGGGGGGPSTTYGAPNGGGNGYSRPSSTYGTPSTGGGSFGGSGGYSGGGGGYSGGGNGYSGGGGGGYSGGNGGGYSGGGNGGGYSGGNGGGYSGGGGGGYSGGGGGGYSGGGNGYSGGGGGGYSGGNGGYSGGNGGYSGGGGGYSGGGGG GQSYASNGGYQY 17Nasonia RPEPPVNSYLPPGQGGGFGGGRPSGASPSDQYGPPDFQGAGGRGGQAAGGN vitripennisFGGGGNGFGGAPSSSYGPPGFGSNEPNKFSGAGGGGAGRPQDSYGPPAGGNGFAGSAGAGNSGRPGGAAAGGRPSDSYGPPQGGGSGFGGGNAGRPSDSYGPPSAGGGGFGGGSPGGGFGGGSPGGGFGGGNQGAPQSSYGPPASGFGGQGGAGQGRPSDSYGPPGGGSGGRPSQGGNGFGGGNAGRPSDSYGPPAAGGGGFGGNAGGNGGGNGFGGGRPSGSPGGFGGQGGGGRPSDSYLPPSGGSGFGGGNGRQPGGFGQQGGNGAGQQNGGGGAGRPSSSYGPPSNGNGGGFSGQNGGRGSPSSGGGFGGAGGSPSSSYGPPAGGSGFGNNGGAGGRPSSSYGPPSSGGNGFGSGGQGGQGGQGGQGGRPSSSYGPPSNGNGGFGGGNGGRPSSNGYPQGQGNGNGGFGGQGGNGGRPSSSYGPPGGDSGYPSGGPSGNFGGSNAGGGGGGFGGQVQDSYGPPPSGAVNGNGNGYSSGGPGGNGLDEGNDEPAKYEFSYEVKDDQSDGRKQIVEYEADQDGFKPQIRYEGEANTGAGGAGGYPSGGGGDSGYPSGPSGAGGNAGYPSGGGGGAGGFGGNGGGSNGYPSGGPSGGQGQFGGQQGGNGGYPSGPQGGSGFGGGSQGSGSGGYPSGGPGGNGGNNNFGGGNAGYPSGGPSGGNGFNQGGQNQGGSGGGYPSGSGGDAAANGGYQYS 18 NasoniaRAEAPISGNYLPPSTSYGTPNLGGGGGGGGGFGGGAPSSSYGAPSSGGGFG vitripennisGSFGGGAPSSSYGAPSTGGSFGGGAPSSSYGAPSSGGSFGGSFGGGAPSSSYGAPSFGGNAPSSSYGAPSAGGSFGGGAPSNSYGPPSSSYGAPSAGGSFGGSSGGSFGGSFGGGAPSSSYGAPAPSRPSSNYGAPSRPSSNYGAPSSGGSGFGGGSGFGGGRPSSSYGAPSSGSFGGGFGGGAPSSSYGAPAPSRPSSNYGAPAPSRPSSNYGAPAPSRPSSSYGAPSRPSSNYGAPSRPSSNYGAPSSGGSGFGGGSGFGGGRPSSSYGAPSSGSFGGGFGGGAPSSSYGAPAPSRPSSNYGPPSSSYGAPSSGGSGGFGGGAPSSSYGAPSFGGSSNAVSRPSSSYGAPSSGGG QSYASNGGYQY 19Pediculus EPPVKTSYLPPSASRSLNSQYGAPAFTDSNELVAPSPNSNFHDSYNQQQQS humanusFDLSNGLSVPSAAGRLSNTYGVPSAQGANVPSFDSSDSIAVDAAGRSGNSF corporisSSHVPSSTYGAPGNGFGGGSRSSQSGAPSSVYGPPQARNNNFGNGAAPSSVYGPPQARNNNFGNGGAPSQVYGPPKARNNNFGNGAAPSSVYGPPQARNNNFGNGAAPSSVYGPPQARNNNFANSAAPSQVYGPPQARNNNFGNGAAPSSVYGPPQSSSFSSPSGRSGQLPSATYGAPFERNGFGSQGSSGFQGYEPSKRSQTTEDPFAEPAKYEYDYKVQASDETGTEFGHKESRENESARGAYHVLLPDGRMQIVQYEADETGYRPQIRYEDTGYPSAASSRSNNGFNGYQY 20 AnophelesKREAPLPPSGSYLPPSGGAGGYPAAQTPSSSYGAPTGGAGSWGGNGGNGGR gambiae str.GHSNGGGSSFGGSAPSAPSQSYGAPSFGGQSSGGFGGHSSGGFGGHSSGGH PESTGGNGNGNGNGYSSGRPSSQYGPPQQQQQQQSFRPPSTSYGVPAAPSQSYGAPAQQHSNGGNGGYSSGRPSTQYGAPAQSNGNGFGNGRPSSSYGAPARPSTQYGAPSAGNGNGYAGNGNGRSYSNGNGNGHGNGHSNGNGNNGYSRGPARQPSQQYGPPAQAPSSQYGAPAQTPSSQYGAPAQTPSSQYGAPAQTPSSQYGAPAQTPSSQYGAPAPSRPSQQYGAPAPSRPSQQYGAPAQTPSSQYGAPAQTPSSQYGAPAQTPSSQYGAPAQTPSSQYGAPAQQPSSQYGAPAPSRPSQQYGAPAQQPSAQYGAPAQTPSSQYGAPAPSRPSQQYGAPAQAPSSQYGAPAPSSQYGAPAQQPSSQYGAPAQTPSSQYGAPSFGPTGGASFSSGNGNVGGSYQVSSTGNGFSQASFSASSFSPNGRTSLSAGGFSSGAPSAQSAGGYSSGGPSQVPATL PQSYSSNGGYNY 21Glossina RPEPPVNTYLPPSAGGGSGGGSPLAPSDTYGAPGVNGGGGGGGGPSSTYGA morsitansPGSGGGNGNGGGGFGKPSSTYGAPGLGGGGNGGGRPSETYGAPSGGGGNGFGKPSSTYGAPNGGGGNGGPGRPSSTYGAPGSGGGNGGSGRPSSTYGAPGLGGGNGGSGRPSSMYGAPGLGGGNGGSGRPSSTYGAPGSGGGNGGSGRPSSTYGAPGSGGGNGGSGRPSSTYGAPGNGNGGNGFGRPSSTYGAPGSGGSNGNGKPSSTYGAPGSGGGGGRPSDSYGPPASGNGGRNGNGNGQSQEYLPPGQSGSGGGGGYGGGSGSGGSGGGGGGGYGGDQDNNVVEYEADQEGYRP QIRYEGDGSQGGFGGDGDGYSYEQNGVGGDGGGAGGAGGYSNGQNLGANGYS SGRPNGGNGGGRRGGGGGGGGSGGGQNLGSNGYS SGAPNGFGGGNGQGYSGGRSNGNGGGGGGRNGGRYRNGGGGGGGRNGGGSNGYNYDQPGSNGFGRGGGNGENDGSG YHY 22Atta cephalotes RSEPPVNSYLHPGSDTSGTNGGRTDLSTQYGAPDFNNRGNGNSGATSFGGSGAGNGPSKLYDVPIRGNTGGNGLGQFRGNGFESGQPSSSYGAPKGGFGENRGNRGRPSTSYGVPDSNRNNRGGFGNGGSEARPSTSYGVPGANGNQGGFGSGSIGGRPSTSYGVPGANGNNGDSFRNGDIGGRPSTNYGAPGANGNHGGGNGGNGRPSNNYGVPGANGNTNGKGRLNGNSGGGPSNNYGSPNGFGKGLSTSYGSPNRGGNDNHYPSRGSFINGGINGYSSGSPNGNAGNFGHGDESFGRGGGEGENTGEGYNANAQEESTEPAKYEFSYKVKDQQTGSDYSHTETRDGDHAQGEFNVLLPDGRKQIVEYEADQDGFKPQIRYEGEANADGGYGSGLNDNNDGYSSGRPDSESGGFANSGFNGGSSNGGYPNGGPGERKLGGFNNGGSSGYQSGRSAGQSFGRDNAGDLNNDIGGYFSNSPNNIGDSDNANVGSNRQNDGNSGYQY 23 AnophelesKREAPLPPSGSYLPPSGGGGGGGGYPAAQTPSSSYGAPAGGAGGWGGNGNG darlingiNGNGNGGRGGYSNGGGHSGSAPSQSYGAPSAPSQSYGAPSQSYGAPAAAPSQSYGAPSFGGNGGGASHGSGGFTGGHGGNGNGNGYSSGRPSSQYGPPQQQQQPQQQSFRPPSTSYGVPAAPSSSYGAPSANGFSNGGRPSSQYGAPAPQSNGNEFGAPRPSSSYGAPSRPSTQYGAPSNGNGNGYAGHGNGNGHGNGNGHSNGNGNGYNRGPARQPSSQYGPPSQGPPSSQYGPPSQYGPPSSGTSFIAYGPPSQGPPSSQYGAPAPSRPSSQYGAPAQTPSSQYGAPAQTPSSQYGPPRQSSPQFGAPAPRPPSSQYGAPAQAPSSQYGAPAQTPSSQYGAPAQAPSSQYGAPAPSRPSSQYGVPAQAPSSQYGAPAQAPSSQYGAPAQTPSSQYGAPSFGSTGGSSFGGNGGVGGSYQTASSGNGFSQASFSASSFSSNGRSSQSAGGYSSGGPSQVPATIPQQYSSGGGSYSSGGHSQVPATLPQQYSSNGGYNY 24 AcromyrmexRSEPPVNSYLPPGPGTSGANGGQTDLSIQYRASDFNNRGNVNGNSGATSFG echinatiorGPGASNGPSKLYDVPIGGNAGGNGLGQFRGNGFEGGQPSSSYGAPNGGFGENRGNGGKPSTSYGVPDSNGNNRGGFGNGGSEGRPSTSYGLPDASRNNGNGFGNVGNEDKPSTNYGIPANGNKVSGFGNVGSEGRPSTSYGVPGANGNQGFGSGGIGGRPSTSYGVPGVNGNNGGGFENVGRPSTSYGTPDARGNNGGSFRNGDIGGRPSTNYGIPGANGNHGGGNGGNGRPSSNYGVPGGNGNTNGKGRFNGNSGGRPSNSYGSPNGFGKGLSTSYSPSNRDGNGNHYPSGDSNRGSFVNGGINGYPSGSPNGNAGNFRHGDESFGRGGEGGGRSTGEGYNANAQEESTEPAKYEFSYKVKDQQTGSDYSHTETRDGDHAQGEFNVLLPDGRKQIVEYEADQDGFKPQIRYEGEANADGEYDSGGLNDNNDGYSSGRPGSESGGFANNSGFNGGSSNGGYPSGGSGEGKLGFNSGGNSGYQSGRPAGQSFGRDNAGDLSNDIGGFSNSPNNIGGDNANVGSNRQNGGNSGYQY 25 AcyrthosiphonESPYGGGSSNSNGNGRNGGYGGKGQYGGGNGGGVGSSSASPFFSGANQYGS pisumQSGLSGAANNRYPSFGSKFGGNKGSYGGSSSRNNGRYGSGSASGYGSGSSGGLGSTGRSTGGYGGGSSGSYGSGSSGSLGSSTGSNGIYGAGSSGGFGSGSSGSYGGGSSGGFGSGSSGSYGGGSSGGFGSGSSGSYGGGSSGGFGSGSSGSYGGGSSGGFGSGSSGNYGSGSSGSYGSGGGGLGGASSGNNDGYGAGGSGSYDQLGGANGNGLGGSGNDPLSEPANYEFSYEVNAPESGAIFGHKESRQGEEATGVYHVLLPDGRTQIVEYEADEDGYKPKITYTDPVGGYAGDRQSGNSYGGNGGFGGSGSLGGSGGNLGGLYNGGGSSNNGAGYGGSSSSLGSRYGGSGGSSGSGVGGGYGGSGSSSGGIGSSYGGSGSLSGGLGGGYGGSGSSSGGLGGGYGGSGGSSGGGFGGLGGSGGSSGSGYGGSGSSSGGLGNSYGGSGSSNGGLGGGYSGSGGSSGGLGGGYGASSGSSGSGLGGGYGGSGSSSGGLGSGYGGLGSSSGGLGGGYGGSGSSSGGLGGGYGGSGSSNGGIGGGYGGSSGSSGGLGGGYGGSGSSSGGLGGGYGGSGGSNSGLGSSYGGSGSTNGGLGGGYGGLGSSSGGLGGGYGGSGGSNGGIGGGYGGSSGSGGSQGSAYGGSGSSSGSQGGGYGGSGSSSGGLGGGYGSSSGSSSGLGGSYGSNRNGLGSGSSYS 26 DrosophilaRPEPPVNSYLPPSPGDSYGAPGQGQGQGQGGFGGKPSDSYGAPGAGNGNGN virilis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rosophilaRPEPPVNSYLPPSDSYGAPGQSGPGGRPSDSYGAPGGGNGGRPSDSYGAPG erecta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utzomyiaRPEPPANTYLPPSSSYAAPGQQGGSGFGGGGGSGGSGGFGQPGAFGRPSSS longipalpisYGPPSQGGAGGGFGSDSQFGGGFGGGAGGFGSGGSGAPGASQRPSSSYGPPGQTGGGGFGAQGAPGSSFGPGGGFGGGSPGQAGSPGFQRPSSSYGPPGQSPGGGFSQQGGAPGASQRPSSTYGAPGQGAGGFGQGGSGGFGGTGGSVAIGGRPSSSYGAPGQGSSGGFGGGSGGFGSQAPSTSYGAPGQGSPGGGFGSQGGPGGQPGSPGFGGSQRPSSSYGPPGQGGAPGQGGSPGFGASSRSGGAGGFGASQQPSSSYGPPGQGAGSGFQGTGGGFGGPGQRPGFGGSQTPATSYGAPGQAGGASGGFGGAGAQRPSSSYGPPGQASGFGGGSSGGGFGGGSSGGFGGNQGGFGGNQGGFGGSQTPSSSYGAPSFGSGGSPGAAGGAGGFGQGGVGGSGQPGGFGGGDQGYPPRGGPGGFGPGSGGSGAGGPIAGGSGSGYPGGSDSGSNEPAKYDFSYQVDDPASGTSFGHSEQRDGDYTSGQYNVLLPDGRKQIVEYEADLGGYRPQIKYEGGSSGGAGGYPSGGPGSQGGAGGYPSGGPGGPGSPGGAGGYQSGAAGGAGGYPSGGPGGPGAGGYPSGGPGGPGSQAGGFSGGFGGGSDGAFGGAGGFSQGGAGGGDAGYPRGGPGGFGGAGSPGFGGSGSPGFGGSGSPGAQGSSGFGGTGGGFGGGADGYPRGGPGAGQSGFQDGRGATGGAGQPGGRGSFGRPGSARGGSSSNGYANGGAEGYPRDNPQNRGSGYS 29 Rhodnius prolixusKRDDPLRRFLAPLVGGGNGSGGGGGGYNYNKPANGLSLPGGGGALPPATSYGVPDRPAPVPSSPPSSSYGAPQPSPNYGAPSSSYGAPSQQPSRSYGAPSQGPSTSYSQRPSSSYGAPAPQTPSSSYGAPAQQPSGSYGAPSGGGGSSGYTGGAQRPSGSYGAPSQGGPSGNYGPPSQQPSSNYGAPSQTPSSNYGAPAQRPSTSYGAPSQPPSSSYGSPPQRASGYPSSSSGPSNGYSPPAQRPSSSYGPPSQQPASSYGAPSQTPSSNYGPPAPIPSSNYGAPSQPPSKPSAPSSSYGTPSQTPSTSYGAPSQAPSSSYGAPSRPSPPSSSYGAPSQGPSSSYGPPSRPSQPSSPSSGYGAPSQGPSSSYGAPSRPSSPSSSYGAPPSSSYGAPSRPSPPSSSYGAPSQGPSSSYGPPSRPSQPSSPSSGYGAPSQGPSSSYGAPSRPSSPSSSYGAPPSSSYGAPSRPSPPSSSYGAPSQGPSSSYGPPSRPSQPSSPSSSYGAPSQGPSSSYGAPSRPSPPSSSYGAPSQGPSSSYGPPSRPSQPSSTYGVPSGGRPSTPSSSYGAPPQALSSTYGAPSGRPGAPSQKPSSSYGAPSLGGNASRGPKSSPPSSSYGAPSVGTSVSSYAPSQGGAGGFQSSRPSSSYGAPSTGPSSTYGPPSQPPSSSYGVPSQPPSSNYGVPSQGVSGSVGSSSPSSSYGAPSQIPSSSYGAPSQSSIGGFGSSRPSSSYGAPPQAPSSSYSAPLRAPSTSYGAPSGGSGSNFGSKPSTNYGAPSQPPSTNYGPPSQPPSSSYGTPSRAPSPTYSTPQSSGTSFGSRPSSSYGVPSQPTTNYGAPSQTPSSNYGAPPASSAPSSTYGRPSQSPSSSYGAPSPSSSSSSYESPSQPPSSSYGAPSQGPSSSYGAPSRPSSTYGAPSPSSPSTNYGAPAPSSNYGTPAQDLTGSYAAPSQPPSAGYGAPSGQPSSGGKQNFQVKNPFAGQTHQVYPAVSSISFGLPSQSFNTAIQGQEPSQSYGAPTASSPSSSYGAPTGTGSSQPGQSYASNGGYSYS 30 Rhodnius prolixusQPPFNHYLPAARGSGSNSAQYTAPSSKFGTSTGQYGQPPSEVPRGLQQGSYAEDVHSSRSVNPSSQNGIPSGHFSSLSSNYGAPSSDYSRSFLRYGTLSNKYGVPNSALGSLSSRNNKTPATQLSYQPSSHYDSRSTSEDQFISSRVSDSQYGASSVRRFLPSSQYSTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSQYGTPSSPPSQYGGPYSMRTSAPNSQYGTPSSFRTSPSSQFGSSSAHSSSLSKFRSVPSSPYGTLSAIRSTHSSQYGTPSSFSDSTSSSHNGLPSHYPGSGFSGSSVNDQKSYTGNVFGQSHSRVANGDQHARSYTLAGGNEISEPAKYDFNYDVSDGEQGVEFGQEESRDGEETNGSYHVLLPDGRRQRVQYTAGQYGYKPTISYENTGTLTTGRQQFSNGFYNVQQSGSESQEHLGRSTGQNSYGGSNGYESGVGYQSGVGRRSRPAGSY 31 Solenopsis invictaRSEPPINSYLPPRAGSSGANGGRTDLTTQYGAPDFNNGGGATSFSGNGAGDGPSKLYDVPVRGNAGGNGLGRGNGFGGGQPSSSYGAPNGGSNENRGNGGRPSTSYGVPGANGNNGGGFGNGGDKGRPSTSYGVPDASGSSQGSFGNVGNGGRPSTNYGVPGANGNGGGFGNAANEGKPSTSYGVPGANGNSQGGFGNGGRPSTGYGVPGANGNNGGGFGGRPSTSYGAPGANGNHRGGNGGNASPSTNYGVPGGNNGNTNGKGRFNGGNSGGGPSNNYGVPNENAFGGGLSTSYGPPSRGGNGNSGYPSGGSNGGSFVNNGANGYPSGGPNGNAGNFGDGRGGKGGGSSGEGYNDNAQEGSTEPAKYEFSYKVKDQQTGSEYSHTETRDGDRAQGEFNVLLPDGRKQIVEYEADQDGFKPQIRYEGEANAGGGYSSGGSNDNNDGYSSGRPGSEAGGFANNSGFNGSGTNGGRSSGGPGDGNPGGFNSGGGGGYQSGRPAGQSFGRDNDGGLSGDIGGYFANSPSNNIGGSDSANVGSNRQNGGNGGYQY 32 CulexKREAPLPGGSYLPPSNGGGAGGYPAAGPPSGSYGPPSNGNGNGNGAGGYPS quinquefasciatusAPSQQYGAPAGGAPSQQYGAPSNGNGGAGGYPSAPSQQYGAPNGNGNGGFGGRPQAPSQQYGAPSNGNGGARPSQQYGAPNGGNGNGRPQTPSSQYGAPSGGAPSSQYGAPSGGAPSQQYGAPNGGNGNGRPQTPSSQYGAPSGGAPSQQYGAPNGGNGNGRPQTPSSQYGAPSGGAPSSQYGAPSGGAPSSQYGAPAGGAPSSQYGAPAGGAPSSQYGAPAGGAPSSQYGAPAGGAPSSQYGAPAGGAPSSQYGAPSSQYGAPAGGAPSSQYGAPAGGAPSSQYGAPSGGAPSSQYGAPSGGAPSSQYGAPAGGAPSSQYGAPSGGAPSS 33 BactroceraRPEPPVNSYLPPSANGNGNGGGRPSSQYGAPGLGSNSNGNGNGNGGGRPSS cucurbitaeQYGVPGLGGNGNGNGNGGGGGRPSSSYGAPGLGGNGNGNGNGGGRPSSQYGVPGLGGNGNGNGNGNGGGRPSSTYGAPGLRGNGNGNGNGNGRPSSTYGAPGLGGNGNGNGNGNGRPSSTYGAPGLGGNGNGNGNGNGRPSSTYGAPGLNGNGLGGGQKPSDSYGPPASGNGNGYSNGGNGNGNGGGRPGQEYLPPGRNGNGNGNGGRGNGNGGGANGYDYSQGGSDSGESGIVDYEADQGGYRPQIRYEGEANNGAGGLGGGAGGANGYDYEQNGNGLGGGNGYSNGQDLGSNGYSSGRPNGNGNGNGNGNGNGYSGRNGKGRNGNGGGQGLGRNGYSDGRPSGQDLGDNGYASGRPGGNGNGNGGNGNGYSNGNGYSNGNGNGTGNGGGQYNGNGNGYSDGRPGGQDNLDGQGYSSGRPNGFGPGGQNGDNDGNGYRY 34 TrichogrammaRPEPPVNSYLPPGQGGQGGFGGSGGRPGGGSPSNQYGPPNFQNGGGQNGGS pretiosum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rosophilaQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechelliaGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGSAGRPSSSYGAPGSGPGGRPSDSYGP 36 DrosophilaQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechelliaGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGSAGRPSSSYGAPGSGPGGRPSDSYGP 37 DrosophilaQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechelliaGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGSAGRPSSSYGAPGSGPGGRPSDSYGP 38 DrosophilaQSGAGGRPSDTYGAPGGGNGGRPSDSYGAPGQGQGQGQGQGGYGGKPSDSY sechelliaGAPGGGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGGNGNGGRPSSSYGAPGQGQGNGNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNNGGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGSGGRPSSSYGAPAQGQGGFGGRPSDSYGAPGQNQKPSDSYGAPGSGNGSAGRPSSSYGAPGSGPGGRPSDSYGP 39 DrosophilaYSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechelliaPGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGNGGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 40Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechelliaPGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGNGGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 41Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechelliaPGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGNGGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 42Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechelliaPGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGNGGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 43Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechelliaPGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGNGGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 44Drosophila YSSGRPGNGNGNGNGGYSSGRPGGQDLGPSGYSGGRPGGQDLGAGGYSNVK sechelliaPGGQDLGPGGYSGGRPGGQDLGRDGYSGGRPGGQDLGAGAYSNGRPGGNGNGGSDGGRVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSSGRPGGRPGG NGQDSQDGQ 45 3X FLAGGDYKDDDDKDYKDDDDKDYKDDDDK 46 Alpha matingMRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVA factor precursorVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR protein sequence 47 EA repeat EAEA 48Linker SG

1. A method for producing a composition comprising a recombinant resilin protein, comprising: culturing a population of recombinant host cells in a fermentation, wherein said recombinant host cells comprise a vector comprising a secreted resilin coding sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 7, 8, 9, 10, 11, and 12, and wherein said recombinant host cells secrete a recombinant resilin protein encoded by said secreted resilin coding sequence; and purifying said recombinant resilin protein from said fermentation.
 2. The method of claim 1, wherein said recombinant resilin protein is a full-length or truncated native resilin.
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. The method of claim 1, wherein the recombinant resilin protein comprises an alpha mating factor secretion signal.
 7. The method of claim 1, wherein the recombinant resilin protein comprises a FLAG-tag.
 8. The method of claim 1, wherein the vector comprises more than one secreted resilin coding sequence.
 9. The method of claim 1, wherein the recombinant host cells are yeast cells.
 10. The method of claim 9, wherein the yeast cells are methylotrophic yeast cells.
 11. (canceled)
 12. The method of claim 1, wherein the recombinant host cells produce the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.
 13. The method of claim 1, wherein the recombinant host cells produce a secreted fraction of the recombinant resilin that is greater than 50% as compared to the total recombinant resilin protein expressed by the recombinant host cells.
 14. The method of claim 1, wherein the recombinant host cells secrete the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.
 15. The method of claim 1, wherein greater than 80% of the recombinant resilin is outside of the recombinant host cells in said fermentation.
 16. The method of claim 1, wherein the fermentation comprises at least 2 g recombinant resilin/L.
 17. The method of claim 1, wherein purifying said recombinant resilin protein comprises generating a first pellet fraction and a first supernatant fraction by centrifuging the fermentation; and isolating the recombinant resilin protein from the first pellet fraction.
 18. The method of claim 17, wherein purifying said recombinant resilin protein further comprises: adding a chaotrope to the first pellet fraction to generate a solution in which the recombinant resilin protein is soluble; generating a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction comprising said chaotrope; and isolating the soluble full-length resilin from the second supernatant fraction.
 19. A vector comprising a secreted resilin coding sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 7, 8, 9, 10, 11, and
 12. 20.-35. (canceled)
 36. A recombinant host cell comprising the vector of claim
 19. 37.-43. (canceled)
 44. A fermentation comprising the recombinant host cell of claim 36 and a culture medium suitable for growing the recombinant host cell. 45.-47. (canceled)
 48. A composition comprising recombinant resilin derived from a fermentation of claim
 44. 49.-67. (canceled)
 68. The fermentation of claim 44, wherein the recombinant host cell secretes recombinant resilin at a rate of at least 2 mg/g dry cell weight/hour.
 69. A composition comprising a gel comprising a recombinant resilin selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and
 12. 70. The composition of claim 69, wherein the recombinant resilin comprises the sequence set forth in SEQ ID NO:
 1. 71. The composition of claim 69, wherein the recombinant resilin comprises the sequence set forth in SEQ ID NO:
 4. 72. The composition of claim 69, wherein the gel comprises full-length resilin.
 73. The composition of claim 69, wherein the gel comprises a plurality of cross-linked recombinant resilins.
 74. The composition of claim 73, wherein the cross-linking comprises enzymatic cross-linking, photochemical cross-linking, or chemical cross-linking. 